lIlITHWIT!IiMillllllllfilflflilillll'lllIll!Ill I 3 1293 10586 6341 This is to certify that the . dissertation entitled COUPLING BETWEEN ELECTRON TRANSPORT AND ATP FORMATION IN ISOLATED CHLOROPLASTS presented by J. Michael Gould has been accepted towards fulfillment of the requirements for flow“ @041 Major professor \ Date W3 3 M5 U is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LlBRARlES _—. » RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. .fn _, (g . W298: f7 / hi... . _‘ 4;- a r COUPLING BETWEEN ELECTRON TRANSPORT AND ATP FORMATION IN ISOLATED CHLOROPLASTS By J. Michael Gould A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1982 ABSTRACT COUPLING BETWEEN ELECTRON TRANSPORT AND ATP FORMATION IN ISOLATED CHLOROPLASTS By J. Michael Gould l. The photochemical reduction of lipophilic electron accep- tors such as oxidized pfphenylenediamines or pfbenzoquionones by chloroplasts is coupled to phosphorylation with an efficiency (P/ez) approximately one-half that observed when hydrophilic acceptors such as ferricyanide or methylviologen are reduced. The reduction of lipophilic acceptors is largely insensitive to the plastoquinone antagonist dibro- mothymoquinone (DBMIB) as is the associated phosphorylation. This obser- vation shows that the lipophilic acceptors are reduced by electrons from water at or before the point of involvement of plastoquinone in the electron transport chain. It also shows that there is a site of coupling of electron transport to ATP formation before the involvement of plasto- quinone. 2. At concentrations somewhat higher than those required to inhibit electron transport, DBMIB itself behaves as a lipophilic electron acceptor. However, DBMIB seems to be reduced via a pool of electrons (plastoquinone?) whereas other lipophilic acceptors seem to be reduced before the electrons from the photosystem II units are pooled. This J. Michael Gould conclusion is based on the fact that DBMIB reduction is exceptionally sensitive to DCMU inhibition at low light intensities whereas the re- duction of other lipophilic acceptors is exceptionally sensitive to DCMU at all light intensities. 3. It is possible to insert electrons into the electron transport chain at a point after the site of inhibition by DBMIB by using appropriate exogenous donors such as diaminodurene, diamiotoluene and reduced 2, 6-dichlorophenolindophenol while using methylviologen as the electron acceptor. The reactions depend entirely on photosystem I. The transfer of electrons from these exogenous donors to methylviologen is also associated with phosphorylation, very likely via the coupling site known to be between plastoquinone and cytochrome f, 4. By using lipophilic electron acceptors in the presence of DBMIB one can isolate the functions of photosystem II and the associated phosphorylation reaction (coupling site II). By using exogenous electron donors such as diaminodurene in the presence of DCMU one can isolate the functions of photosystem I and the associated phosphorylation reaction (coupling site I). The efficiency of coupling site II is pH-independent over the range 6-9 (0.3-0.4 ATP molecules per pair of electrons). In contrast, the somewhat higher efficiency of coupling site I is strongly pH-dependent (0.6 at pH 8, §_0.l at pH 6.5). Moreover, the energy trans- fer inhibitor HgCl2 is a much less effective inhibitor of site II phos- phorylation. Electron flow through the less efficient site II is inde- pendent of the presence or absence of phosphorylation or of uncouplers, whereas electron flow through coupling site I is greatly increased by phosphorylation or uncouplers. J. Michael Gould 5. Over a wide pH range the sums of the efficiencies of the two coupling sites measured separately as above are very close to the efficiencies obtained at the same pH's when they are operating simulta- neously,as in the overall Hill reaction. This implies that the charac- teristics of the coupling sites are not altered by the presence of the added inhibitors, donors and acceptors. 6. During the reduction of DBMIB by photosystem 11 there is an uncoupler-sensitive uptake of protons by the chloroplasts which is reversed when the reduction ceases. About 0.5 protons are reversibly taken up per electron transported over the pH range 6-8.5. The extent of the uptake decreased 40-60% during phosphorylation. This is consistent with the notion that proton gradients are used for phosphorylation as postulated in the chemiosmotic hypothesis. 0n the other hand, the site- specific inhibitions by Hg++ and low pH are difficult to accomodate with- in the concept of a delocalized trans-membrane gradient as the driving force for phosphorylation. Such site-specificities can be reconciled with a modified version of the chemiosmotic hypothesis. The modified version specifies that protons accumulate inside the membrane, that the resulting strictly local increase in proton activity drives phosphoryla- tion, and that this local proton activity can equilibrate with a trans- membrane gradient. A postulated difference in the hydrophobicity of the microenvironments of the two coupling reactions could then account for the observed site-specificities. to my wife, Brenda ii ACKNOWLEDGEMENTS I would like to express my appreciation and gratitude to Dr. Norman E. Good and Dr. Seikichi Izawa for their invaluable advice, enduring patience and wise counsel. I am also grateful to Dr. Good for his scholarly insights into matters sometimes outside the realm of basic science. I Thanks are also due to my fellow graduate student and good friend, Dr.Donald Ort, for a fruitful collaboration. Special thanks is due to my understanding wife, Brenda, for her patience and moral support during the preparation of this manu- script. The work described here was supported by the National Sci- ence Foundation of the United States through grants GB 22657 and GB 37959X to Drs. Izawa and Good. TABLE OF CONTENTS Page LIST OF TABLES ........................................ ........... vi LIST OF FIGURES ........................................, ......... viii LIST OF ABBREVIATIONS ............................................ xiii INTRODUCTION ..................................................... l A. Electron Transport Pathways ................................. 2 B. Coupling "Sites" and the Hypotheses for the Mechanism of Coupling Between Electron Transport and ATP Formation ..... 9 C. Stoichiometry Between Electron Transport and ATP Formation, and the Location of the Coupling Sites ...................... ‘2 .EXPERIMENTAL METHODS ............................................. 15 A. Preparation of Chloroplasts ................................. -l7 B. Measurement of Electron Transport ........................... 18 C. Actinic Light ............................................... 22 D. Measurement of ATP Formation ................................ 22 E. Measurement of Changes in H+ Contentration (pH Changes) ...... 25 F. Inhibitors and Reagents ..................................... 26 RESULTS .......................................................... 28 A. Determination of a Second Coupling Site in Isolated Chloroplasts .............................................. 29 B. Functional Separation and Characterization of the Two ATP-Generating Coupling Sites ............................. 36 C. The Relation of Light-Induced Proton Fluxes to the Electron Transport and ATP Formation Associated with Coupling Site II .................................................. 41 DISCUSSION ......................................... ............. 51 A. Functional Separation of the Two Coupling Sites in Chloroplasts ............................................ 52 B. Differences in the Properties of the Two Coupling Sites .... 53 C. The Coupling Mechanism and Coupling Site-Specificity ....... 58 LITERATURE CITED ................................................ 6l APPENDICES ...................................................... 68 Appendix 1: Electron Transport and Photophosphorylation in Chloroplasts as a Function of the Electron Acceptor III. A Dibromothymoquinone-Insensitive Phosphorylation Reaction Associated with Photosystem II ............................. '70 iv Appendix II: Photosystem II Electron Transport and Phosphory- lation with Dibromothymoquinone as the Electron Acceptor... Appendix III: Studies on the Energy Coupling Sites Of Photophosphorylation 1. Separation of Site I and Site II by Partial Reactions of the Chloroplast Electron Trans- port Chain ................................................ Appendix IV: Studies on the Energy Coupling Sites of Photo- phosphorylation III. The Different Effects of Methy- lamine and ADP plus Phosphate on Electron Transport Through Coupling Sites I and II in Isolated Chloroplasts... Appendix V: Site-Specific Inhibition Of Photophosphoryla— tion in Isolated Spinach Chloroplasts by Mercuric Chloride .................................................. Appendix VI: Studies on the Energy Coupling Sites of Photo- phosphorylation IV. The Relation of Proton Fluxes to the Electron Transport and ATP Formation Associated with Photosystem II ........................................... Appendix VII: The Phosphorylation Site Associated with the Oxidation of Exogenuous Donors of Electrons to Photosystem I ............................................ Appendix VIII: Electron Transport Reactions, Energy Con- servation Reactions and Phosphorylation in Chloro- plasts ................................................... Page 81 90 104 ll6 120 I37 152 Table II. II. III. II. LIST OF TABLES APPENDIX I The effect Of dibromothymoquinone on electron transport and photophosphorylation in chloroplasts with different electron acceptors ........................................ . Inhibition of various reactions in Chloroplasts by dibromothymoquinone and KCN ............................... APPENDIX II Effect of photosystem I inhibitors on electron transport and photophosphorylation with various electron acceptors.... The lack of effect of phosphate, ADP or uncouplers on dibromothymoquinone reduction ............................. APPENDIX III Effects of reduced DCIP plus ascorbate and 2,5-dimethyl- benzoquinone on post- illumination ATP formation (XE) ...... Effect of KCN treatment on electron transport and phosphorylation associated with the photosystem I dependent reaction reduced DCIP -> methylviologen ......... Effect of phosphorylating conditions on electron trans- port as a function of the electron acceptor ............... APPENDIX V Effect of HgCl on photophosphorylation in spinach chloroplasts with various electron acceptors .............. Effect of HgClz on postillumination ATP formation (XE) ..... vi Page 74 78 85 85 96 97 99 118 118 Table Page APPENDIX VI I. Stoichiometry between proton uptake and electron transport (HT/6') with dibromothymoquinone or methy- lviologen as electron acceptor ............................. 130 II. Effect of phosphorylation and arsenylation on the extent of the light-induced proton uptake associated with the electron transport pathway H20 —9 photosystem II —> dibromothymoquinone .................................. l3l APPENDIX VII 1. Diaminodurene, diaminotoluene, and reduced 2, 6-dichlorophenolindophenol as donors of electrons to photosystem I ........................................... 144 APPENDIX VIII I. Phosphorylation efficiency as a function of the donor of electrons to Photosystem II ....................... 159 vii Figure l. The conventional "Z-scheme" of non-cyclic electron transport in spinach chloroplasts ......................... 2. Scheme for cyclic electron transport in spinach Chloroplasts .............................................. 3. Sites of inhibition of non-cyclic electron transport in Spinach chloroplasts ................................... 4. Post-illumination ATP formation (XE) associated with electron transport through coupling sites I and II ........ 5. A comparison between the efficiencies of proton uptake and phosphorylation associated with each of the two coupling sites in chloroplasts ............................ 6. Electron transport pathways and phosphOrylation sites in chloroplasts ........................................... APPENDIX I 1. Effect of dibromothymoquinone on electron transport (E.T.) and phosphorylation (ATP) with ferricyanide (FeCy) or oxidized p-phenylenediamine (PDox) as electron acceptor .................................................. 2. Effect of dibromothymoquinone on electron transport and phosphorylation with ferricyanide and methylviologen (MV) as electron acceptors ................................ 3. Effect of higher concentrations of dibromothymoquinone on electron transport and phosphorylation with ferricyanide or methylviologen as acceptors ............................ 4. Effects of dibromothymoquinone on digitonin-treated chloroplasts .............................................. 5. Simplified scheme of the electron transport pathways, LIST OF FIGURES phosphorylation reactions and inhibition sites ............ viii Page 44 48 54 75 75 76 76 80 APPENDIX II Figure 1. The effect of KCN on endogenous and dibromothymoquinone- catalyzed oxygen uptake (Mehler reaction) in illuminate chloroplasts ............................................ Effect of dibromothymoquinone on electron transport and phosphorylation in the absence of added electron acceptor .... ............................................ Effect of pH on dibromothymoquinone reduction and associated phosphorylation .............................. Inhibition by dichlorophenyldimethylurea of electron transport with various acceptors ........................ The effect of light intensity on dichlorophenyldimethy- lurea inhibition of electron transport supported by various acceptors ....................................... Effect of light intensity on dichlorophenyldimethylurea inhibition of dimethylbenzoquinone reduction ............ A simplified model of electron transport in chloroplasts APPENDIX III Effect of pH on the rates of electron transport and phosphorylation associated with various electron donor- acceptor systems .............. . ......................... Effect of pH on the phosphorylation efficiency (P/ez) of three different electron donor-acceptor systems .... Effect of the energy transfer inhibitor 4'-deoxyphlorizin on electron transport and phosphorylation associated with different donor-acceptor systems ................... Light-induced pH rise in the medium ("proton uptake") associated with the reduction of dibromothymoquinone by photosystem II .......................................... A scheme for electron transport pathways in isolated chloroplasts showing the two sites of energy coupling (~) ..................................................... ix Page 84 84 85 86 86 87 88 94 95 98 100 102 APPENDIX IV Figure Page 1. Effect of the plastoquinone-antagonist dibromothy- moquinone on electron transport and ATP formation associated with the photoreduction of ferricyanide and oxidized p-phenylenediamine by isolated chloroplasts ......... 108 2. Effect of the electron transport inhibitors dibromothymoquinone and KCN on electron transport (E.T.) and phosphorylation (ATP) when dimethquuinone is the electron acceptor ........................................ 109 3. Effect of the uncoupler methylamine hydrochloride on electron transport (E.T.) and ATP formation when ferricyanide (FeCy) and oxidized p-phenylenediamine (PDox) serve as electron acceptors ........................... 110 4. Effect of methylamine on the rate of electron transport when ferricyanide (FeCy) and oxidied p-phenylenedia- mine (PDox) are the electron acceptors ....................... llO APPENDIX V 1. Effect of HgCl on electron transport (E.T.) and phosphorylatiofl associated with various electron transport pathways ........................................... ll8 APPENDIX VI 1. Light-induced pH changes associated with the partial electron transport pathway H O —> photosystem II -> dibromothymoquinone in isolateg chloroplasts ............ l25 2. Recorder tracing of the apparent kinetics Of the reversible pH rise associated with Photosystem II electron transport from water to dibromothymoquinone ......... 126 3. Initial kinetics of the light induced pH rise and electron transport measured under both flash and continuous illumination with dibromothymoquinone as the l28 electron acceptor ............................................ 4. Comparison between the initial rates of proton uptake and electron transport for the complete electron trans- port chain (H20-—> Methylviologen) and the photosystem II partial reaction H20 -—>dibromothymoquinone .................. 129 APPENDIX VI (continued) Figure 5. Effect of phosphorylation on the extent of the light- induced proton uptake associated with electron transport from water to dibromothymoquinone ............ Effect of arsenylation on the extent of the light induced proton uptake associated with electron trans- port to dibromothymoquinone ..................... ...... APPENDIX VII Effect of bovine erythrocyte superoxide dismutase on the electron transport and phosphorylation associated with the diaminodurene —> methylviologen reaction ...... Effect of diaminodurene (DAD) concentration on the rate of electron transport and phosphorylation in the diaminodurene —> methylviologen reaction ............... Double reciprocal plot showing the effect of diaminodurene concentration on electron transport in the diaminodurene —> methylviologen reaction ........... Effect of pH on the rate of electron transport and phosphorylation associated with the diaminodurene 7? methylviologen reaction ............................ a) Effect of DCIPH concentration on electron transport and pgosphorylation in the DCIPH2 -—> methylviologen reaction ........................ b) Double reciprocal plot of the data presented in (a) c) Replot of some of the data presented in (b) showing the component of the DCIPH —> Page ...... 132 ...... 133 ....... 142 ...... 143 ...... 144 ...... 145 ...... 146 methylviologen reaction with the higher apparent A“ ....... 146 Effect of the energy transfer inhibitor HgCl on electron transport and phosphorylation assoc1ated with the diaminodurene —>methylviologen reaction ...... A scheme for electron transport pathways in isolated chloroplasts showing the two Sites of energy transduction ( ~ ) .............................. xi 147 ...... 149 Figure 1. APPENDIX VIII Chloroplast reactions currently available for the study of photosynthetic electron transport and phosphorylation .......................................... Reduction of a lipophilic strong oxidant with electrons from water ..................................... A conventional chemiosmotic interpretation of phosphorylation in chloroplasts .......................... The oxidation of catechol and ferricyanide by hydroxylamine-treated Chloroplasts with methylviologen as electron acceptor ...................... Phosphorylation as a function of the time of illumination ............................................. Photophosphorylation efficiency as a function of the external pH .............................................. A comparison of the efficiencies of proton uptake and ATP synthesis at different pH's ...................... A modified model of the chemiosmotic mechanism which allows for site specificity in the utilization of the energized state for phosphorylation ...................... xii Page 153 156 157 158 160 161 162 164 ADP ATP CF cyt DAD DAT DBMIB DCIP DCMU DMQ EDAC Fecy H+/e' MV PC PD P/e2 PSI PSII LIST OF ABBREVIATIONS adenosine diphosphate adenosine triphosphate coupling factor one cytochrome diaminodurene diaminotoluene 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (dibromothymoquinone) 2,6-dichlorophenolindophenol 3(3,4-dichlorophenyl)-l, l-dimethylurea 2,5-dimethyl-p-benzoquinone l-ethyl-3-(3-dimethylaminopropyl) carbodiimide ferricyanide; Fe(CN)g' ratio of the number of protons translocated per electron transported methylviologen plastocyanin p-phenylenediamine ratio of the number of ATP molecules synthesized per pair of electrons transported inorganic phosphate photosystem I photosystem II xiii quencher; primary electron acceptor for photosystem II primary electron acceptor for photosystem I xiv INTRODUCTION INTRODUCTION The single most important biological process, upon which all life on earth ultimately depends, is the process of photosynthesis, for it is this process which converts the electromagnetic radiation of sun- light into the chemical bond energy required by all living systems to maintain their entropy gradient with the environment. The mechanisms involved in this energy conversion are incompletely understood, but it is known that at least two distinct processes are involved: i) the use of light quanta to transfer electrons and thereby store energy in oxi- dized and reduced products; ii) an associated conversion of some of the light energy into the chemical bond energy of ATP. This dissertation deals primarily with studies concerning the nature of the latter process. A. Electron Transport Pathways In green plants, the energy of the quanta absorbed by the photo- synthetic pigments is ultimately transferred to the Chlorophyll embedded in the chloroplast membranes where it is used to move electrons to or- bitals which represent higher energy levels. The excited electrons are then transferred to alowpotential electron acceptor, and the oxidized Chlorophyll which is generated is subsequently reduced by electrons com- ing from water. There are two such photoreactions occurring in chloro- plasts. These photoreactions are arranged in series, and are connected by a sequence of stepwise oxidation-reduction reactions mediated by a variety of electron carriers (Figure 1). One photochemical reaction 7/ (volts) 0 /§ / CYI f‘PC U +0.5 - ‘ Chi 0]: (P700) Pizfl) J +1.0 "' 0 cm On 2 (P680) Figure l. The "2 Scheme" for Non-cyclic Photosynthetic Electron Transport in Isolated Chloroplasts. (photosystem II) results from the excitation of a special type of Chlorophyll a_molecule (P690) and causes the sequential accumulation of four electron holes (for four quanta absorbed) in an as yet unidentified primary electron donor (Z), which is subsequently reduced by the four electrons generated by the simultaneous oxidation of two molecules of H20 to produce molecular oxygen (02). The primary acceptor of electrons from the Photosystem II reaction (Q; E6 E -100 mV) is also as yet un- identified, although it has been suggested to be a quinone-type compound (Stielh and Witt, 1969). Electrons from Q are rapidly transported to a substituted quinone, plastoquinone (E5 2 50 mV), which is present in large excess over the other electron transfer components (excluding chlorophyll). This suggests that plastoquinone may represent an electron pool between the two photochemical reactions. There is strong kinetic evidence to support such a concept (Kok gt_al,, 1969). The oxidation of plastohydroquinone by a c-type cytochrome (cytOChrome f, E6 = 340 mV) is believed to be one of the sites along the electron transport chain where the formation of ATP is coupled to the exergonic redox reactions (see be- low). Reduced cytochrome f_transfers electrons to the reaction center Chlorophyll of Photosystem I (P700; E6 5 495 mV; absorption max. = 700 400 mV). II? nm) via a copper containing protein known as plastocyanin (E6 The primary electron acceptor for photosystem I (X; E6 < -550 mV) is also unidentified, although there is some reason to believe that it may be a bound ferredoxin. In intact chloroplasts, the electrons from X are carried via a series of carriers to NADP+ (Figure 1). However, in the Chloroplast preparations utilized in this study, the outer envelope and most if not all of the soluble or very weakly bound enzymes are lost, 5 including the electron carriers from X to NADP+. For this reason it is necessary to introduce into the reaction system an artificial electron acceptor so that ongoing electron transport may be studied. The ability of the Chloroplast to catalyze the reduction of artificial oxidants, known as the Hill reaction, has provided an extremely valuable-tool for the investigation of electron transport pathways and ATP formation. Ex- amples of the artificial acceptors of electrons from X used in this study are methylviologen and ferricyanide. The light-driven transport of electrons through the linear transport chain (Figure 1), known as non-cyclic electron transport,is stoichiometrically coupled to the synthesis of ATP (non-cyclic photo- phosphorylation). It is also possible, by the addition of an appropriate exogenous electron carrier, to observe photophosphorylation which is not coupled to the net flow of electrons from water to some acceptor."Cyclic" photophosphorylation, which is mediated by photosystem I, involves the shuttle of electrons from X (see Figure 1) back to some component of the linear electron transport chain via the exogenous carrier. That is, the exogenous carrier (e.g. pyocyanine, phenazine methosulfate) is reduced by X and subsequently reoxidized by another carrier, probably cytochrome f_ (see Appendix VII). There is some evidence that a b-type cytochrome (cytochrome b563) may also participate in such cyclic electron flow (Figure 2). In order to study the number of "sites" of coupling between ATP formation and electron transport it was necessary to operate selected portions of the non-cyclic electron transport chain. In order to do this, one must specifically inhibit the flow of electrons through certain A " X l A‘\ CYI b563(?) ”m; Figure 2. Cyclic Electron Transport Pathway in Chloroplasts. (”A" represents the exogenously added electron transport mediator.) regions of the chain, and at the same time insert or extract electrons from particular carriers in the chain. The electron transport inhibi- tors available include compounds or treatments which will block the oxidation of water, the oxidation of O, the oxidation of plastohydroqui- none, the reduction of cytochrome f and the flow of electrons through plastocyanin (Figure 3). When this study was initiated, however, the ability to specifically insert and extract electrons at desired loca- tions was not well established, and one important objective of the work was to define more precisely the regions where exogenous oxidants and re- ductants interact with the electron transport Chain. Saha gt 31, (1971) were the first to point out that, of the exogenous electron acceptors aVailable, hydrophilic oxidants (Class I acceptors) such as ferricyanide and methylviologen are reduced almost ex- clusively by photosystem I, while strong lipophilic oxidants such as p: phenylenediimine and prbenzoquinone (Class III acceptors) are reduced primarily by photosystem II. Inhibitor studies (reviewed in Appendices VII and VIII) have substantiated this conclusion. (Class II acceptors (e.g., indophenols) have been defined as acceptors which uncouple phos- phorylation from electron transport). The site where some reduced compounds donate electrons to the chain has remained unclear for nearly a decade. Compounds such as diaminodurene and reduced dichlorophenolindophenol supply electrons to the transport chain at some point after plastoquinone, so the oxidation of these compounds requires only photosystem I (Izawa gt al., 1966). It has also remained uncertain where mediators of cyclic electron transport donate electrons to the transport chain. .mumm_qoco_;u cw “commence cocuumpm UWPuxu-:oz Co mLngnw;:_ mzowcm> Co cowuu< mo mmuwm .m mczm_m __ . >§A1Hmdtlo=dllx 30.9: = GAIL—immdizlom: zox 93m 9.28 :28 zomzz B. Coupling "Sites" and the Hypotheses for the Mechanism of Coupling Between Electron Transport and ATP Formation A major aspect of the work described in this dissertation deals with determining the stoichiometric relationship between phosphorylation and electron transport in chloroplasts, and in locating the particular electron transfer sequences associated with this coupling phenomenon. It thus becomes important to define exactly what a "coupling site" is. Unfortunately, there is no precise definition available since there is substantial controversy as to the molecular events involved in the mechanism of the coupling process itself. Depending upon the coupling hypothesis, at least three different definitions of a "coupling site" are possible. 1) Chemical coupling. Simply stated, this hypothesis (Slater, T953) proposes that certain carriers in the electron transport chain interact directly with an as yet unidentified compound to produce a high- energy chemical intermediate. The subsequent hydrolysis of this inter- mediate by an ATP synthetase is coupled to the condensation of ADP and inorganic phosphate to make ATP. (The enzyme responsible for ATP syn- thesis in chloroplasts has been identified as a 325,000 MW multisubunit protein known as coupling factor one (CF1), bound to the surface of the thylakoid membrane.) According to the formulations of the chemical coupling hypothe- sis, a coupling "site" represents an electron transfer step which results in the formation of a high-energy chemical intermediate. However, as yet there is no direct evidence for the existence of such an inter- mediate, although the absence of evidence does not necessarily 10 constitute evidence for absence. Indeed, it is quite conceivable that a high energy chemical intermediate may be in the form of a charge trans- fer complex or similar entity which cannot be isolated. 2) Chemiosmotic coupling. This hypothesis, largely based on the ideas of Mitchell (1961, 1966) saggests that the electron carriers are arranged in the membrane in such a manner that the reduction by an electron of a hydrogen carrier near the outer surface of the membrane re- sults in the uptake of a proton, which is subsequently released to the inside of the thylakoid by the oxidation of the hydrogen carrier by an electron carrier near the inner surface of the membrane. This electron transport dependent uptake of protons results in the formation of a pH gradient across the thylakoid. Furthermore, any lag in the movement of charge compensating counterions would result in an electrical potential across the membrane as well. According to this hypothesis, the pH gradi- ent and membrane potential constitute a high-energy state of the membrane which can be discharged by a reversible, proton pumping ATPase (CF]) to make ATP. By this model, any electron transport step which results in the deposition of protons in the inner phase of the thylakoid can be consid- ered a coupling site. There is an impressive amount of evidence which lends strong support to the chemiosmotic hypothesis. Chloroplasts do accumulate pro- tons in the light in an electron transport-dependent reaction (Neumann and Jagendorf, 1963). Furthermore a pH gradient produced across the chloroplast membrane, either in the light by electron transport or in the dark by an acid treatment, is capable of driving phosphorylation (Hind and Jagendorf, 1965; Jagendorf and Uribe, 1966). Perhaps the most 11 convincing evidence comes from the elegant experiments of Racker and Stoeckenius (1974), who employed a simplified system containing bacteri- orhodopsin (which catalyzes a light driven proton pump in phospholipid vesicles) and purified mitochondrial hydrophobic proteins and ATPase in- corporated into phospholipid vesicles. In the light these vesicles supported substantial rates of phosphorylation even though no electron transport components were present. 3) Conformational Coupling. This hypothesis is less well defined than the other two major hypothesis presented above. It postu- 1ates that exergonic electron transport reactions give rise to energeti- cally unstable conformations of membrane proteins. The relaxation of these metastable conformations is coupled to the synthesis of ATP. Thus, accordingtn this model, a coupling site would be any electron transport step giving rise to a conformational Change capable of driving phosphory- lation. Actually, there is good evidence that conformational changes in the ATP synthesizing enzyme (CF1)are involved in energy conserva- tion, although the exact nature of the involvement is still unclear. En- ergization of chloroplasts induces a conformational change in CF1 which results in the exposure of 50-100 previously buried tritium exchange sites to the aqueous phase (Ryrie and Jagendorf, 1971, 1972). Similar conditions also expose previously concealed sulfhydryl and lysine resi- dues to attack by Nfethylmaleimide (McCarty gt_al,, 1972) and trinitro- benzenesulfonic acid (Oliver and Jagendorf, 1975), respectively. In general, regardless of which of these hypotheses one champ- ions, a coupling site is defined as an area in the electron transport 12 sequence which gives rise to an energy-rich intermediate or state which can be discharged to make ATP. A coupling site is distinct from a phos- phorylation site, which is defined as the particular location where the high-energy intermediate or state is enzymatically utilized to make ATP (i.e. the CF1 molecule). C. Stoichiometry Between Electron Transport and ATP Formation, and the Location Of the Coupling Sites. The quantitative relationship Of electron transport to ATP formation can be expressed as the phosphorylation efficiency, or P/e2, which is defined as the ratio of ATP molecules synthesized to pairs of electrons passing through the coupling site. Although it is known that at least 1.5 molecules of ATP per 2 electrons are required for the CO2 fixation reactions, the exact stoichiometry of non-cyclic electron trans- port has been disputed for some time, with some investigators arguing for a theoretical P/e2 of only 1.0 (see Avron and Neumann, 1968). However, one of the reasons for the low P/e2 values observed was the use of suboptimal experimental conditions. With better chloroplast preparations and im- proved buffers, Winget gt_al, (1966) were able to Obtain P/e2 ratios con- sistently and significantly greater than 1.0. This suggested that more than one coupling site may be associated with the non-cyclic electron transport chain. Other indirect evidence supporting this conclusion was supplied by Izawa gt_al, (1966), and Izawa and Good (1968), who showed that under cer- tain conditions a correction of the electron transport rate for the rate of "basal" electron flow, (i.e. electron flow in the absence of phosphate) gave "corrected" P/e2 ratios of close to 2.0. A large number of alter- nate hypothesis (see Avron and Neumann, 1968, for a review) postulating 13 separate Sites for cyclic and non-cyclic phosphorylation, hidden cycles, and even stoichiometries as high as P/ez = 4 (Lynn, 1967) further con- fused the issue. However, very little gigggt_evidence for more than one Site emerged for nearly a decade. An important breakthrough in this area came with the work of Saha £3 31, (1971), who showed that lipophilic strong oxidants such as p-phenylenediimines and p-benzoquinones are reduced by chloroplasts in a reaction coupled to phosphorylation with an efficiency consistently about one-half the efficiency observed when convential hydrophilic Hill oxidants (e.g. methylviologen) are reduced. They concluded that these lipophilic acceptors were being reduced at a point between two coupling sites. Ouitrakul and Izawa (1973) subsequently showed by inactivating plasto- cyanin with KCN that photosystem I was required for the reduction of hy- drophilic oxidants but not for the reduction of lipophilic oxidants. This provided strong circumstantial evidence for the existence of at least two coupling sites associated with the complete non-cyclic chain. However, direct confirmation of this notion requires a knowledge of the exact location of the coupling sites in the electron transport chain. Evidence for the location of one of the coupling sites comes from studies of the steady—state redox levels of the electron carriers under various conditions. This approach is based on the fact that, in the complete Hill reaction, the rate of electron transport during phos- phorylation is considerably higher than the rate in the absence of phos- phorylation. When the phosphorylation reaction is uncoupled from elec- tron transport , the highest rates of electron transport are observed. These facts have been taken to mean that the formation of a high-energy 14 intermediate or an energized state regulates the rate of electron flux through the coupling site by creating a "back-pressure" on the forward phosphorylating reactions. Thus, in the absence of an uncoupler the electron carriers on the photosystem II side of the rate-limiting coupl- ing site should be more reduced, and those on the photosystem I side more Oxidized, than when an uncoupler is added (to remove the rate-limitation at the coupling site). Using this technique, Kok _e_i_:_ 11. (1969) determined that the rate-limiting step of the Hill reaction came after the electron pool at plastoquinone. Avron and Chance (1966), Larkum and Bonner (1972) and Izawa (1968) similarly showed that the rate-limitation occurred before the reduction of cytochrome f, and Bohme and Cramer (1972) demonstrated directly the existence of a phosphorylation-dependent electron transport reaction between plastoquinone and cytochrome f, Ouitrakul and Izawa's experiments with KCN cannot completely eliminate the possibility that Class III acceptors are reduced both be- fore and after a single coupling site located between plastoquinone and cytochrome f, since the cyanide inhibition site is at a point in the electron transfer chain after cytochrome f_(see Figure 3). One of the objectives of this study was to demonstrate unequivocally the existence of a second coupling site associated with the non-cyclic electron trans- port chain in chloroplasts. The introduction by Trebst's group (Trebst, gt_al,, 1970) of a new inhibitor (dibromothymoquinone; DBMIB) which blocks electron transport at plastoquinone (i.e. bgf95g_the known coupling site) made available a new approach to the problem. Indeed, it was found that DBMIB completely inhibited phosphorylation and electron transport 15 dependent upon the reduction of hydrophilic acceptors, while only slightly affecting the electron transport and phosphorylation associated with the reduction of lipophilic acceptors. This observation provided another, more conclusive demonstration of a second coupling site in Chloroplasts. One of the other Objectives of this study was to find suitable reactions using defined portions of the electron transport chain so that the individual coupling sites could be functionally separated and investigated without resorting to physical disruption of the chloroplast. In the course of studying these "partial" reactions it was observed that the characteristics of the two coupling sites are substantially different. This was a particularly intriguing development since any theory of the coupling mechanism must accomodate the observed differences. Finally, the stoichiometric relationship between proton uptake, electron transport and ATP formation associated with the newly isolated coupling site was studied in detail in an attempt to elucidate the role of protons in the energy conservation mechanism. EXPERIMENTAL METHODS EXPERIMENTAL METHODS A. Preparation of Chloroplasts The chloroplasts employed for the research described in this thesis consisted of intact, naked lamellae. That is, during the isola- tion procedure the outer chloroplast membrane was stripped away, and enzymatic activities attributable to soluble stroma enzymes were lost. However, the lamellae retained their general morphological appearance, the grana remaining largely intact. Chloroplasts were isolated from leaves of fresh market spinach (Spinacea oleracea L.) in the cold (4°C). Chilled leaves were washed in cold distilled water before being ground briefly (3-7 seconds) in a Waring Blender. The grinding medium consisted of 0.3 M NaCl, 30 mM N-tris(hydroxymethyl)methylglycine (tricine) -Na0H (pH 7.8), 3mM MgCl2 and 0.5 mM ethylenediaminetetracetic acid (EDTA). After filtering the homogenate through multiple layers of cheesecloth (prerinsed with dis- tilled water), the chloroplasts were sedimented by centrifugation at about 2500 x g for two minutes. The pellet was resuspended in a medium con- taining 0.2 M sucrose, 5 mM Nez-hydroxyethylpiperazine-Nf-2-ethanesulfonic acid (HEPES)-NaOH (pH 7.5), 2 mM MgCl2 and 0.05 percent bovine serum a1- bumin. Whole cells and debris were removed by a brief (30-45 second) cen- trifugation at 2000 x g. Chloroplasts in the resulting supernatant were sedimented at 2000 x g for four minutes, resuspended in a fresh volume of 17 18 the same medium, and again sedimented. The final pellet was taken up in a small volume of the suspending medium. In some cases, when the pres- ence of metal chelating agents had to be avoided, the EDTA and bovine serum albumin were omitted from the grinding and suspending media, res- pectively, while the tricine and HEPES buffers were replaced by 2LN7tris (hydroxymethyl)methylJaminoethanesulfonic acid (TES) and Nr2-hydroxye- thylpiperazine-N73-propanesulfonic acid (HEPPS) buffers respectively. The concentration of chlorophyll present in the final stock chloroplast suspension was determined spectrophotometrically by the method of Arnon (1949). A 0.1 ml aliquot of the chloroplast suspension was di- luted to 10 ml with 80 percent (v/v) acetone-water. After centrifugation at 5000 x g for seven minutes to remove insoluble chloroplast residues, the absorbance (A) of the green supernatant was measured at 663, 652 and 645 nm. The concentration of chlorophyll was determined using the equation 100 ([8.02(A663) + 20.2(A645)] + [29(A652)1 ) = 2 ugrams chlorophyll/ml stock suspension. Stock suspensions of chloroplasts were routinely adjusted to 1600-2000 ug chlorophyll/m1 and stored in an ice bath. Aliquots of this stock suspension were further diluted with suspending medium before being added to the reaction mixtures. 8. Measurement of Electron Transport The light-induced flux of electrons through the chloroplast electron transport chain was measured by one of several methods, depending upon the experimental conditions and the nature of the electron acceptor 19 employed. These methods are described in detail below. 1) Potassium ferricyanide as electron acceptor. When ferricyan- ide (E6 = 420 mV) served as the acceptor of electrons from photosystem I, electron transport was generally measured spectrophotometrically. This was accomplished by measuring the decrease in the absorbance of the sample of 420 nm in either a Bausch and Lomb Spectronic 505 spectro- photometer or a spectrophotometer consisting of a Beckman model DU mono- chrometer equipped with a photomultiplier and associated log conversion electroniCs. Both spectrophotometers were modified to permit illumina- tion of the sample cuvette (l x l x 4 cm) by an actinic light beam at right angles to the measuring beam. The photomultipliers were protected from stray light by filters which blocked all of the scattered red-orange actinic light (eg. Baird Atomic interference filter; peak transmittance 420 nm, band-width lOnm). The rate of reduction of ferricyanide was de- termined by continuously recording the decrease in absorbance at 420 nm on a strip chart recorder with a time-base drive. The number of equival- ents of ferricyanide reduced was calculated assuming a millimolar ex- tintion coefficient for ferricyanide of 1.06 at 420 nm. A second method for determining the rate of electron transport when ferricyanide served as the electron acceptor consisted of measuring the rate of oxygen production. Changes in the oxygen concentration of the reaction mixture were determined polarographically using a Clark-type ox- ygen electrode covered with a teflon membrane (Yellow Springs Instrument Co.), in a thermostatted reaction cuvette in which the reaction mixture was stirred continuously by a small magnetic stirring bar. Changes in the current flow through the electrode (which are directly proportional to 20 changes in 02 concentration) were amplified and recorded on a strip chart recorder. The recorder span was calibrated in uequivalents by using a known number of uequivalents of ferricyanide as the electron acceptor and allowing the reaction to continue until all of the ferri- cyanide had been reduced. 2) Methylviologen as electron acceptor. The reduction of the low potential electron acceptor methylviologen (MV; E5 = -446 mV) by photo- system I was followed polarographically as the rate of change Of oxygen concentration in the reaction mixture described in the manner immediately above. However, in the case of methylviologen reduction, the reaction was followed as oxygen uptake rather than as oxygen production, since the reduced methylviologen radical (MV?) is rapidly reoxidized in air. Thus, the reaction proceeds as follows: light / - + H20 chloroplasts > 5 02 + 28 + 2” 2e' + 2MV++ > 2 MVT ++ > H202 + 2MV 2mvt + 02 + 2H+ overall H O + P02 -—-————+> H 0 2 2 2 Therefore the overall rate of oxygen uptake (due to the reduction and subsequent autoxidation of methylviologen) is equal to the rate of oxygen production (one 02 consumed equals 4 electrons transported). However, when 02 evolution by photosystem II is inhibited and an exogenous donor of electrons to photosystem I such as diaminodurene (DAD), diaminotoluene (DAT) or reduced 2, 6—dichlorophenolindophenol (DCIPHZ) is employed, the rate of oxygen uptake due to the reoxidation of reduced methylviologen is 21 twice as great for the same rate of electron transport (one 02 consumed equals two electrons transported) (Izawa gt 21,, 1966). Thus, for the transport of two electrons, the reaction becomes: light + - AHZ chloroplasts > A + 2H + 29 2e' + 2MV++ ———> 2va + + ++ _ 2MV. + 02 + 2H ———-—-—> 2MV + H202 overall AH2 + 02 > H202 + A where AH2 represents any one Of the exogenous electron donors listed above. 3) Lipophilic (Class III) oxidants as electron acceptors. Lipophilic oxidized compounds such as p-phenylenediimines or lipophilic p-benzoquinones will serve as electron acceptors, being reduced primarily at a point in the electron transport chain between photosystem II and Photosystem I (Saha gt_al,, 1971). These compounds were added to the re- action mixture in the reduced form and subsequently oxidized by the addi- tion of an excess of ferricyanide. Thus, when the Class III acceptor was reduced by chloroplasts in the light, it was immediately reoxidized by the excess ferricyanide present in the reaction mixture. Therefore it was possible to measure electron transport as ferricyanide reduction either spectrophotometrically or as oxygen evolution. (See ferricyanide reduction above.) When dibromothymoquinone served as the Class III acceptor, it was also possible to measure electron transport as the indirect reduction of ferricyanide as described above or, above pH 8.0, as oxygen uptake 22 (see methylviologen reduction), since the reduced dibromothymoquinone was rapidly reoxidized by molecular oxygen at pH >8. C. Actinic Light. Illumination of the reaction mixture was with a 500 W slide projector without the focusing lens. The beam was passed through a one liter round-bottomed flask filled with a dilute (approxi- mately 2%) CuSO4 solution. This functioned both as a condensing lens and as a heat filter. The beam was then passed through a red-orange filter (transmittance > 500 nm) and an I-69 Corning heat filter before impinging on the reaction vessel. Reactions were run in small cuvettes maintained at 18°C by a thermostated circulating water bath. The final reaction mixture volume was generally 2 ml. The light intensity at the surface of the reaction cuvette was 400-700 kergs - cm.2 - sec']. 0. Measurement of ATP Formation The rate of photophosphorylation was measured dirctly as the 32 incorporation of P-labelled orthophosphate into AT32P. The reaction mixture contained excess amounts of both radioactive orthophosphate and ADP (5 mM and 0.75 mM respectively). After illumination, a 1.0 ml aliquot of the reaction mixture was pipetted into a 20 x 150 mm test tube and immediately frozen in the dark. 32 The AT P present in the frozen sample was determined by a modification of the method of Neilsen and Lehninger (1955). This techni- 32 que is based on the removal of unreacted P-inorganic phosphate from the sample as phosphomolybdic acid. Saha and Good (1970) have shown that virtually all of the nonextractable radioactivity remaining is incorpor- ated into AT32P. To the frozen 1.0 ml aliquot of the reaction mixture 23 was added 10 ml of lll perchloric acid saturated with a 1:1 isobutanol- toluene mixture. This resulted in a precipitation of the chloroplast proteins, and prevented any further biological reactions from occurring. In order to improve phase separation, 1.2 ml Of acetone was added to the perchloric acid sample mixture. Ammonium molybdate, 1.0 ml of a 10% (w/v) solution was then added . The resulting molybdic acid reacts in 32P-labelled orthophosphoric acid the aqueous solution with the excess to form yellow phosphomolybdic acid. This complex is extremely soluble- in organic solvents, and was removed from the perchloric acid phase by adding 7 m1 Of 1:1 isobutanOl-toluene (saturated with llN perchloric acid). The phases were mixed briefly using the up and down motion of a glass stirring rod which had been flattened at theend. The mixture was then allowed to stand for about one minute. After this time the phases were mixed for about 60 seconds by the piston action of the flattened stirring rod. After the phases had been allowed to separate, the upper (organic) phase containing the phosphomolybdic acid was carefully re- moved using suction on a Pasteur pipet connected to a trap. The remaining (aqueous) phase was gravity-filtered through 9 cm Whatman #‘4filter paper pre-wetted with 0.5 ml distilled water. This filtration removed precipi- tated proteins and remainingtraces of the organic phase. The filtered perchloric acid phase was extracted a second time using 0.1 ml ammonium molybdate and 7 ml isobutanol-toluene (saturated with 1 N perchloric acid). After again removing all traces of the organic phase by suction with a pastuer pipet, an aliquot of the final perchloric acid phase was pipetted into a polyethylene scintillation vial (Packard Instrument Co.) containing sufficient distilled water to make a final volume of 15 ml. 24 The technique used to determine the amount of radioactivity in the perchloric acid extract is based on the method of Gould gt_al_ (1972) and utilizes the phenomenon of Cerenkov radiation. Cerenkov radiation is light emitted by some substances when a particle (such as a high energy B-particle) moves through the substance at a velocity greater than the speed of light in that substance. 32P emits B-particles with sufficeint energy (1.71 MeV) to induce Cerenkov radiation in water (minimum energy required, 0.265 MeV). Thus, it was possible to add an 32P-labelled ATP to aliquot of the perchloric acid extract containing distilled water in a scintillation vial and then measure the Cerenkov radiation with the photomultipliers of a liquid scintillation spectro- meter. The amount of Cerenkov radiation (measured as counts per minute) is proportional to the amount of 32P over a very wide range. Experimental samples (in polyethylene vials) were counted at 4°C for ten minute intervals in a Packard 3003 Series liquid scintilla- tion spectrometer in the coincidence mode. The relative gain and upper and lower window discriminator settings, which were found to give optimum counting efficiency, were 20% and 40-1000 units respectively. Counting efficiency under these conditions was approximately 25-30%. In some of the early experiments, and when the extracted per- chloric acid phase was highly colored, radioactivity was determined in a Geiger-Mefiller immersion tube (20th Century Electronics, Surrey, England) connected to a Nuclear Chicago scaler. Counts per minute were related to ATP by the following calibra- tion procedure: an aliquot of the stock solution of radioactive phosphate was diluted with 0.1 M NazHPO4 to give a solution containing 0.1 umole of 25 the original stock (radioactive) phosphate in each 1.0 ml of the final solution. A 1.0 m1 aliquot of this final solution was then diluted to 13.8 ml with 1N perchloric acid and an aliquot of this "standard" was added to a scintillation vial or the immersion tube and counted as des- cribed above for the experimental samples. E. Measurement Of Changes in H+ Concentration (pH Changes) Light-induced changes in the hydrogen ion concentration in the reaction mixture were detected with a Corning semi-micro ("Tripurpose") combination pH electrode connected to a fast responding Heath/Schlumberger EU 200-30 pH electrometer module equipped with a Heath/Schlumberger EU 200-02 DC offset module to facilitate scale expansion. The output from the electrometer was recorded (using a preamplifier with a gain of 10) on either a Heath/Schlumberger programmable strip chart recorder or on an Esterline Angus strip chart recorder. The response half-time for the pH measuring system was 0.5-1.0 sec. Changes in pH were normally moni- tored with a scale expansion of 0.1 pH unit full scale (10 inches) on the recorder. The noise level was less than 0.002 pH units. In routine pH experiments reactions were run in a final volume of 2.0 ml in thermostatted vessels at 18°C with continuous stirring. Prior to illumination the reaction mixture was adjusted to the desired initial pH with small volumes of dilute NaOH or HCl. Actinic illumination was sup- plied by a 500W slide projector as described earlier. The light intensity was approximately 700 Kergs - cm'2 - sec'1 (ca. 600-700 nm). At the end of each experiment the pH changes registered on the Chart paper were translated into H+ equivalents by back-titrating the reaction mixture in 26 the light (Polya and Jagendorf, 1969) with a known amount of 0.001 M HCl. The overall sensitivity of the pH measuring system was such that 0.5-2.5 mM buffer Could be included in the reaction mixture without ob- scuring the pH change. F. Inhibitors and Reagents Plastocyanin was inactivated by treating chloroplasts in the dark with 30 mM KCN (buffered at pH 7.8) at 0°C for 90 minutes as des- cribed by Ouitrakul and Izawa (1973). The inhibition was checked by de- termining the rate of methylviologen reduction as described above. The inhibitory plastoquinone analog 2.5-dibromo-3-methy1-6- isopropyl-pfbenzoquinone (dibromothymoquinone; DBMIB) was prepared by bromination of thymoquinone in water and was recrystallized several times from hot ethanol. This synthesis was kindly performed by Mr. Peter Felker. Stock solutions were prepared by dissolving dibromothymoquinone in ethanol- ethylene glycol (1:1, v/v). The dihydrochloride salts of diaminodurene, diaminotoluene and pfphenylenediamine were prepared by dissolving the free bases in warm eth- anol, treating with acid washed Norit A and adding concentrated HCl. After cooling the solution for a few minutes on ice, the white crystals of the dihydrochloride were collected. Fresh, colorless solutions of these compounds were made up in 0.01 N HCl each day. 2,5-Dimethyl-pybenzoquinone was purified by recrystalization from hot ethanol. This compound appeared to be stable when stored at 0°C in the dark. Ethanolic solutions of the dimethquuinone were yellow in color. 27 Sodium 2, 6-dichlorophenolindophenol was dissolved in ethanol, filtered, and the concentration determined from the absorbance at 600 nm using a mM extinction coefficient of 21. Ethanolic solutions of 2,6- dichlorophenolindophenol were further diluted with 1 mM NaZHCO3. 3(3,4-Dichlorophenyl)-l, l-dimethylurea (K and K Laboratories) was recrystallized from ethanol. Stock solutions were made in ethanol, with further dilutions being made with 0.01 M NaCl, which helped to pre- vent absorption of the very dilute inhibitor onto the glass walls of the storage vessel. Superoxide dismutase was prepared from fresh bovine erythro- cytes by the procedure of McCord and Fridovich (1969). The specific acti- vity, assayed as the inhibition of cytochrome g_reduction by xanthine oxidase, was > 3000 units per mg. protein. 32P-labelled orthophosphate ADP was purchased from Sigma. (carrier free) was purchased from International Chemical and Nuclear Corp. (ICN). Buffers were prepared by Dr. N.E. Good. RESULTS RESULTS A. Determination of a Second Coupling Site in Isolated Chloroplasts The characteristics of the reduction of lipophilic (Class III) electron acceptors can best be understood by assuming that they accept electrons from some intermediate carrier which normally transfers elec- trons from photosystem II to photosystem I (Saha et al., 1971). Block- ing the flow of electrons to photosystem I by inactivating plastocyanin using either KCN (Ouitrakul and Izawa, 1973) or poly-L-lysine (Brand §3_ 31,, 1971), does not severely inhibit the reduction of Class III accep- tors even though the reduction of Class I acceptors is completely blocked (Ort £5 21,, 1973). Furthermore, the reduction of Class III acceptors is coupled to phosphorylation with an efficiency (P/e2) of approximately one- half that Observed when ferricyanide or methylviologen are reduced (Saha at El. 1971). These observations strongly imply that the Class III ac- ceptors intercept electrons before the involvement of plastocyanin but after one of the two Sites coupling electron transport to ATP formation. One of the coupling sites in chloroplasts almost certainly lies between plastoquinone and cytochome f_(Avron and Chance, 1966; Kok gt_gl,. 1969; BOhme and Cramer, 1972). Thus, if the oxidation of reduced plasto- quinone is prevented, this coupling site should become inoperative. An experimental test of this argument became available with the introduction by Trebst gt_al,(l970) of a new inhibitor, 2, 5-dibromo-3—methy1-6-isopro- pyl-p-benzoquinone (DBMIB), which was claimed to act as a plastoquinone 29 3O antagonist. At very low concentrations DBMIB blocks all of the transfer of electrons from water to Class I acceptors such as ferredoxin-NADP+ or methylviologen and a large part Of the transfer of electrons from water to ferricyanide (BOhme et al., 1971). It also seems to block the transfer of electrons from cytochrome 6559 to cytochrome f_and it certainly pre- vents the reduction of cytochrome f_by electrons from photosystem II (BOhme and Cramer, 1971). These results lend support to the conclusion of Trebst et_gl, (1970) that DBMIB acts at the level of involvement of plastoquinone in the electron transport chain. 1) The site of DBMIB inhibition and its effects on electron trans- port and_phosphorylation with different electron acceptors. The effects of DBMIB on the reduction of Class I(hydrophilic) and Class III (lipophi- lic) electron acceptors are very similar to the effects of the plasto- cyanin antagonists KCN and poly-L-lysine. The flow of electrons to Class I acceptors such as ferricyanide or methylviologen is almost completely sensitive to the inhibitor, while the flow of electrons to Class III ac- ceptors such as oxidized p-phenylenediamine may be largely insensitive (Appendix I, Fig. l). Regardless of the phosphorylation efficiency (P/ez) associated with the reduction of the Class III acceptor in the absence of the inhibitor, the P/e2 ratio always falls to about 0.4 in its presence (Appendix I, Table I). These results indicate that there is a site of phosphorylation associated with the electron transport pathway before the DBMIB inhibition site. Consequently, precise identification of the inhibition site is a matter of critical concern. Trebst _e_t al_. (1970) proposed that. DBMIB blacks electron transport at the level of plastoquinone, but their evidence was 31 somewhat inconclusive. Indeed, the effects of DBMIB described above are identical to the effects of KCN and poly-L-lysine, which almost certainly act at the level of plastocyanin (Izawa et al,, 1973; Brand et al., 1972b). DBMIB differs importantly from KCN and poly-L-lysine in its effects on photosystem I—dependent reactions supported by exogenous elec- tron donors such as diaminodurene. In the presence of DCMU (to block the flow of electrons from photosystem II), the flow of electrons from dia- minodurene to methylviologen is strongly inhibited by either KCN or poly- L-lysine. In sharp contrast, however, DBMIB has no appreciable effect on the diaminodurene + methylviologen reaction (Appendix I, Table II). It is clear, therefore, that the Site of DBMIB inhibition must come between the point of reduction of Class III acceptors and the site of KCN (or poly-L- lysine) inhibition at plastocyanin. This is entirely consistent with the view that dibromothymoquinone acts as a plastoquinone antagonist. 2) DBMIB as an electron acceptor. Early in the course of these studies it was noted that methylviologen reduction seemed to be more sensitive to DBMIB than was ferricyanide reduction. Furthermore, the phosphorylation efficiency (P/ez) associated with ferricyanide reduction decreased with increasing concentrations of DBMIB to a plateau near P/e2 = 0.4. Indeed, at somewhat higher levels of DBMIB than were required for complete inhibition of methylviologen reduction, ferricyanide reduction 1.mg chlorophyll'], actually increased to a rate .1 300 uequivalents - h' about one-half the original (uninhibited) rate (Appendix 1, Figs. 2-4). Because the P/e2 associated with ferricyanide reduction at high DBMIB concentrations (> 511M) is similar to the P/e2 associated with reduction of Class III acceptors in the presence of KCN, poly-L-lysine or low 32 concentrations of DBMIB (0.51JM), it seems reasonable to conclude that at higher concentrations DBMIB can act both as an inhibitor of the flow of electrons to photosystem I and as a.lip0philic:"Class III" accep- tor ofelectrons from photosystem II. The reduced DBMIB is rapidly reoxi- dized by ferricyanide, but for thermodynamic reasons, not by methylvio- logen. The DBMIB reduced by chloroplasts can also react with oxygen to produce H202 when ferricyanide is not present. However, this reaction requires higher levels of DBMIB (10-201IM), and the reoxidation of re- duced DBMIB by 02 becomes seriously rate-limiting below pH 8 (Appendix II, Fig. 2). In contrast, the reoxidation of reduced DBMIB by ferri- cyanide is not rate-limiting under any of the conditions employed in this study since the rate of DBMIB-mediated ferricyanide reduction is constant with time until virtually all of the ferricyanide has been reduced. For these reasons the DBMIB-ferricyanide system was generally employed in the studies described below. 3) The effect of 3(3,4-dichlorgphenyl) —1, l-dimethylurea (DCMU) on DBMIB reduction. DBMIB reduction resembles the reduction of other Class III acceptors in its insensitivity to inhibitors such as KCN and poly-L-lysine (Appendix 11, Fig. 1, Table l), but differs significantly in its sensitivity to DCMU. DBMIB reduction exhibits about the same sensitivity to DCMU as does the reduction of the Class I acceptor ferri- cyanide. In contrast, other Class III acceptors are extremely sensitive to concentrations of DCMU which have very little effect on ferricyanide reduction (Appendix II, Fig. 4). 33 DBMIB reduction differs from the reduction of other Class III acceptors in another important characteristic. When the light-intensity is made rate-limiting, DBMIB reduction, like ferricyanide reduction, be- comes more sensitive to inhibition by a given concentration of DCMU, while the sensitivity of the reduction of other Class III acceptors is independent of light intensity (Appendix II, Figs, 5,6; Ouitrakul and Izawa, 1973). Again DBMIB behaves more like a Class I acceptor than a Class III acceptor under these circumstances. It should be pointed out that the intensity-dependent and intensity-independent effects of DCMU are not related to the maximum absolute rates of reduction of the elec- tron acceptors in the absence of DCMU and in fact represent an actual dif- ference between the reduction pathways of DBMIB and other Class III ac-‘ ceptors. ' It should also be added that the quantum efficiency of DBMIB reduction (DBMIB = lOiIM) is only about 30% of the normal ferricyanide- mediated Hill reaction (Appendix II, Fig.5). Apparently DBMIB has a rather strong inhibitory effect on the photochemical reactions of photo- system II. 4) The site of DBMIB reduction. The fact that KCN and poly-L- lysine have no effect on the rate of electron transport virtually proves that photosystem I is not involved in the reduction of DBMIB. Moreover, DBMIB itself is a potent inhibitor of the transfer of electrons to photo- system I, probably blocking electron transport at the level of plasto- quinone (see above). It follows that DBMIB must accept electrons either before or at its own site of inhibition. In these respects DBMIB re- sembles the reduction of other lipophilic quinones and quinonediimines 34 (Class III acceptors) which also accept electrons from a carrier close to photosystem 11. However, as shown in the preceding section, the re- duction of DBMIB more closely resembles the reduction of Class I accep- tors in its response to DCMU and light intensity. These differences can be readily explained in terms of the following model. There are good reasons for believing that the photochemistry and much of the associated thermochemistry of photosystem II takes place in independent structural units (Kok et_al,, 1970). In other words, it seems that quanta are converted one by one into charge separations within independent structures. Thus the electrons and holes made available by quantum conversions in one structural unit are not directly available for chemical reactions in other units. While the exact nature of the inhibi- tion by DCMU is not clear, it seems likely that DCMU acts by somehow inactivating photosystem II units, one molecule of inhibitor totally sup- pressing the activity of one unit. A partial inhibition by DCMU probably means that a certain proportion of the photosystem II units have been inactivated. However, there is no reason to suppose that photosystem I must be confined to the same unit structures as photosystem II. Consequently it is not unreasonable to suppose that the electrons generated by photosystem II may be pooled at some step before the reduction of P700. Indeed one might expect reduced plastoquinone to serve as a common electron pool interconnecting electron transport chains on the basis of its chemical na- ture and its abundance. In fact, Siggel et_al,, (1972) and Malkin and Michael (1972) have reached this conclusion from their flash experiments and fluorescence induction studies using chloroplasts poisoned with DCMU. 35 We are now in a position to understand the effect of light intensity on DCMU inhibition. When light is limiting, the activity of photosystem II is presumably also limiting and the inhibition should be strictly pro- portional to the number of units inactivated by DCMU. The same will be true, regardless of light intensity, if the electrons are never pooled. However, if the electrons are pooled and the pooled electrons are utilized by a subsequent rate-determining slow step, the situation is quite differ- ent. Now a smaller number of functioning photosystem II units can keep the slow reactions draining the electron pool saturated. Thus, as the light intensity increases, fewer and fewer photosystem II units are re- quired and the efficacy of a given concentration of DCMU decreases. Such considerations suggest that Class III acceptors are reduced before the electrons from photosystem II are pooled (Ouitrakul and Izawa, 1973). Presumably these membrane-permeating strong oxidants react direct- ly with the photosystem II units. Clearly ferricyanide and other Class I acceptors must be reduced after the electrons are pooled since their re- duction is less sensitive to DCMU at high light intensities. Although DBMIB is highly lipid-soluble and is a moderately strong oxidant, it does not seem to react directly with photosystem II units but rather with the source of pooled electrons. It seems reasonable to conclude, therefore, that DBMIB, by virtue of its structure, reacts in some specific way at the site of plastoquinone involvement in electron transport, accepting elec— trons from reduced plastoquinone and at the same time blocking further transport of electrons to cytochrome f. 36 8. Functional Separation and Characterization of the Two ATP- Generating Coupling Sites. The conclusion seems inescapable that the non-cyclic electron transport pathway in chloroplasts includes at least two energy conserva- tion sites associated with phosphorylation, one located before the plastoquinone electron pool and a second located after plastoquinone but before cytochome f, It has been shown above that it is possible to oper- ate a "partial" electron transport pathway which includes only photo- system II and utilizes only the energy conservation site before plasto- quinone (site II). This is done by using Class III acceptors in conjunc- tion with inhibitors of the flow of electrons to photosystem I (i.e., KCN, poly-L-lysine or DBMIB). Similarly, it is possible to introduce electrons into the transport chain at a point after coupling site II but before coupling site I (See Appendices III,VII and VIII). This is done by using appropriate exogenous electron donors (eg. diaminodurene, diamino- toluene, reduced indophenols) while preventing any contribution of electrons from photosystem II with DCMU. The use of these two types of partial electron transport pathways provides functional separations of the coupling sites so that their characteristics and properties may be studied separately and compared with the characteristics of the overall (Hill) reaction. 1) pH effects. When electrons from water reduce the Class I acceptor methylviologen (MV) through the two coupling sites, both electron flow and phosphorylation Show an optimal pH of approximately 8-8.5. The rate of electron transport in the absence of phosphate (basal rate) is much slower but shows a similar pH optimum (Appendix III, Fig. 1). When 37 diaminodurene (DAD) or reduced 2, 6-dichlorophenylindophenol (DCIPHZ) serves as the donor of electrons to photosystem I in the presence of DCMU (coupling site I only), the effect of pH is very similar to that observed in the HZO-e>MV reaction, with an optimal pH of 8-8.5 and a marked stimu- lation of the rate of electron transport by phosphorylation (Appendix III, Fig. 1 and Appendix VII, Fig. 4). The reductions of the Class III accep- tor 2,5-dimethyl-p:benzoquinone (0M0) or of DBMIB by electrons from water (coupling site II only) exhibit an entirely different effect of pH, how- ever. The pH optimum is considerably mor acidic (7.3-7.8) and the rate of electron flow is the same in the presence and absence of a complete phosphorylation system (see below). A study of the effect of pH on efficiency of phosphorylation (P/ez) associated with each of the electron transport pathways described above revealed some interesting results (Appendix III, Fig. 2, Appendix 11, Fig. 3, Appendix VII, Fig. 4). The P/e2 for the overall reaction (H20 —>MV) is strongly pH dependent, being optimal at pH 8 to 8.5 and falling sharply at lower pH's to a value of about 0.4 at pH 6.5. In contrast, the P/e2 associated with the H20 —>DMQ and H20 —>DBMIB reactions (which utilize only coupling site II) is essentially independent of pH over the range 6.5-9 (P/e2 5 0.4). Thus the pH dependent portion of the P/ezratio for the H20-—>MV reaction must be attributable to a coupling site located after the site of DM0 and DBMIB reduction. In fact, the P/e2 ratios for the DAD-—>MV and DCIPH2->MV reactions are strongly pH-dependent, with an optimum (P/e2 3 0.6) at pH 8-8.5. As the pH is lowered the P/e2 drops sharply to.: 0.1 at pH 6.5. The pH-dependent nature of the phosphoryla- tion associated with the H20—->MV and DAD (or DCIPH2)-—> MV reactions 38 suggests that these reactions may involve the same coupling site. Indeed, the fact that the P/e2 values for the DAD (or DCIPHZ) -9 MV reaction are lower (by about 0.4) than the P/e2 values for the H20 —> MV reaction over the entire pH range tested suggests that a pH independent component of the overall P/ez is missing from DAD (or DCIPHZ) —> MV reaction. If the P/ez) values for the two types of partial reactions (eg. H20 -> DM0 and DCIPH2 —> MV) are added together over the pH range 6-9, the resulting curve is in fact very close to the experimentally obtained curve for the overall reaction H20 —> MV (Appendix III, Fig. 2). This also suggests that the DAD (or DCIPH2)-—> MV reaction is utilizing the coupling Site which normally limits the overall Hill reaction (coupling site I). There- fore, the two types of partial reactions described above seem to provide a reliable and convenient assay for the study of the individual coupling sites. 2) The different effects of ADP plus Pi and uncouplers on electron transport associated with each coupling site. The use of the partial reactions described above led to the observation that the two coupling sites differed in several important characteristics. As has al- ready been pointed out, the rate of electron transport in partial reac- tions which include coupling site I is markedly stimulated by the addition of ADP plus phosphate at pH values higher than 7 (Appendix III, Fig. 1, Table III; Appendix VII, Figs. 2,4), whereas a similar stimulation of electron transport by ADP plus phosphate was not observed in those partial reactions which included only coupling site II (Appendix III, Fig. 1, Table III; Appendix II, Table II; Appendix IV, Figs. 1,2). Apparently the 39 rate of electron flux through coupling site II is not regulated by the "energized state" of the chloroplast. Similar results were obtained when uncouplers such as grami- cidin D or methylamine were added. The rates of electron flow along transport pathways which included coupling site I only or coupling site I plus coupling site II were increased markedly by uncouplers, while the rates of electron flow along transport pathways which included only coupling site II were unaffected by uncouplers (Appendix II, Table II; Appendix IV, Fig. 3). The effects of ADP plus phosphate and of uncouplers are sunmarized in Tablel of Appendix IV. A It is possible to construct a model which explains the differ- ences between the abilities of site I and site II to regulate electron transport. Presumably the stimulation of the rate of electron flow through coupling site I is due to the relaease of "back-pressure" generated against electron flow by the accumulation of the high energy state. Since the potential available between plastoquinone and cytochrome f_(coupling site I) is only about 300 mV, such a "back-pressure" could well slow the flow of electrons (see also discussion). However, a very different situation may be encountered at coupling site II. Here the energy con- serving electron transport reactions may be essentially irreversible be- cause of the large energy input via photosystem II which seems to drive the reaction. Even though a "back-pressure" due to formation of the high energy state existed, the few hundred millivolts involved would be un- likely to effect a significant reversal of the forward reaction which utilizes 1.8 V. 40 3) Effects of energy transfer inhibitors. In view of the results presented in the previous section, an attempt was made to determine if the terminal enzymatic steps of ATP formation supported by each coupling site Showed the same sensitivities to specific inhibitors of the phos- phorylation reaction (energy transfer inhibitors [Good, et a1. 1966 ] ). In the overall reaction H20 —> MV and the partial reaction DCUPH2 —9 MV, the energy transfer inhibitor 4’- deoxyphlorizin (Winget et_gl,, 1969) inhibited both ATP formation and that portion of the electron transport dependent upon phosphorylation in a very similar manner (Appendix III, Fig. 3). However, the inhibitor had no effect on the transport of elec- trons from H20 -->DA0ox either in the presence or absence of ADP plus Pi’ although phosphorylation itself was inhibited. In fact, ATP formation supported by coupling site I and by coupling site II exhibited the same sensitivity to 4' -deoxyphlorizin. Results similar to those described above were also obtained with rabbit antiserum prepared against chloroplast CF1 (Gould, 1975b). Again ATP formation supported by the two coupling Sites showed equal sensitivity to the inhibitor. However, very different results were obtained when yet another chloroplast energy transfer inhibitor, HgClz, was employed. Low concen- trations of HgCT2 (approx. 1 atom Hg++/40 molecules chlorophyll) inhibit ATP formation and phosphorylation-dependent electron transport to a pla- teau of about 50% when the electron transport pathway is from H20 to MV or ferricyanide (Izawa and Good, 1969; Appendix V, Fig. 1). Neither basal (-P1) nor uncoupled electron transport is affected by these low levels of HgClZ. Electron transport and phosphorylation associated with the 41 partial reactions DCIPH2 —> MV and DAD —> MV (coupling Site I only) are Similarly affected by HgC12 (Appendix V, Fig. 1; Appendix VII, Fig. 6). When the electron transport pathway includes only coupling site II, however, entirely different results are obtained. Electron transport and phosphorylation associated with the partial electron trans- port pathway from water to the Class III acceptors PDox’ DADox’ DMQ or DBMIB are completely insensitive to the low levels of HgCl2 which inhibit phosphorylation at coupling site I (Appendix V, Table I, Fig. 1). It is unlikely that this remarkable difference in sensitivity to HgCl2 exhibited by the two coupling sites is an artifact arising from fortuitous reaction conditions when Class III acceptors are employed since the chloroplasts were incubated with HgCl2 for 30 seconds before the addition of the acceptor system. Furthermore, UV spectra of PDox’ DADox’ DM0 and DBMIB remained virtually unchanged in the presence of high concentrations of HgCl2 (33 p M), which means that there was little or no reaction of these substances with the mercury. Moreover, a similar level of inhibi- tion by a given amount of HgClz was obtained at several different concen- trations of the Class III acceptor (Gould, 1975b). C. The Relation of Light-Induced Proton Fluxes to the Electron Transport and ATP Formation Associated with Coupling Site II. The differences between coupling sites I and II described above, that is, the very different degrees of control of electron trans- port by phosphorylation and the very different sensitivities to low pH and mercury, prompted us to undertake further investigations into the mechan- ism by which electron transport may be coupled to phosphorylation at these two sites. The experiments described below deal with a light-induced, 42 reversible proton uptake (i.e., pH rise in the suspending medium) asso- ciated with electron transport through coupling site II, and its re- lation to the mechanism of energy conservation at that site. 1) Light-induced pH rise associated with DBMIB reduction. As shown earlier, the use of substrate concentrations of DBMIB provides a conven- ient reaction system for studying the nature of coupling site II since the function of the substrate as an inhibitor effectively blocks further electron transport to photosystem I and thereby isolates site II from site'I. When a weakly buffered reaction mixture containing chloro- plasts and 20 u M DBMIB was illuminated, a dark-reversible rise in the medium pH was observed (Appendix III, Fig. 4; Appendix VI, Figs. 1,2). Above pH 8.1, where the DBMIB is rapidly reoxidized by molecular oxygen, the pH-rise was observed many times over repeated light-dark cycles. Be- low pH 8, where the reoxidation rate is very slow, the pH shift was main- tained only as long as the reduction of DBMIB continued. As the reduc- tion approached completion and electron transport slowed down, there was a gradual reversal of pH rise even in the light and the pH eventually re- turned to the original level. Subsequent illuminations in the absence of further additions of DBMIB did not restore the pH rise. The uncoupler gramicidin D and the electron transport inhibitor DCMU completely abol- ished the light-induced pH rise. 2) Demonstration of post-illumination ATP synthesis (XE) associ- ated with coupling site II. Hind and Jagendorf (1963) discovered that chloroplasts illuminated in the absence of ADP, Pi and Mg++ generated a capacity to form ATP when ADP, Pi and Mg++ were subsequently added in the 43 dark. This capacity for post-illumination ATP formation (termed XE) has been shown to be closely correlated with the uptake of protons by the chloroplasts in the light (Izawa, 1970). Thus, any reaction causing the reversible uptake of protons by chloroplasts should exhibit post-illumina- tion phosphorylation (XE). Figure 4 shows that the electron transport pathway H20 —>DMQ, which includes coupling site II only (and the associ- ated proton pump - see above) is capable of generating XE. 3) Kinetics and stoichiometry of electron transport and proton gptake(H+/e') at site II. The results presented above (Section C, 1 and 2) are most easily explained in terms of a transmembrane H+ gradient as— sociated with the partial reaction H20 —>photosystem II —> Class III ac- ceptor (eg. DBMIB, 0M0). However, any critical evaluation of the relev- ance of this proton gradient to the coupling mechanism requires a know- ledge of the efficienCy of the proton uptake (H+/e'). Furthermore, since the efficiency of phosphorylation associated with coupling site II is lower than the efficiency of the complete chain where both coupling sites are operating (P/e2 = 0.3-0.4 versus 1.1-1.2,respectively), one might ex- pect that the efficiency of proton accumulation (H+/e') would be corres- pondingly lower when only coupling site II is involved. Because of the technical and/or theoretical problems which have plagued previous attempts to measure H+/e' ratios in chloroplasts (see‘ Appendix VI for a discussion of these problems; see also Jagendorf, 1975), a new technique was developed which gave highly reproducible results and which largely avoided the problems alluded to above. This method was based upon the flash-yield tehnique developed by Izawa and Hind (1967) and involved measurement of the pH changes induced by a series of brief :30 S 1: CL 9. .9 I: C) CD §20 E <12 :63 10 C) E C: “J X 0 Figure 4. 44 — _ 531113 :[I -~:::>”’,/”///’//””’:/ ar’F" _q ’AA/ // ,/snes I + II — ’ I l l 7 7.5 8 pH (dork stage) Post-illumination ATP Formation Capacity (X ) Generated by Electron Transport Through Coupling Sites I and II. (The re- action procedure is given in Appendix 111 except that the electron transport systems were as follows: Site II, H20 —> DMQ; Sites I + II, H20 —> MV.) PLEASE NOTE: Page 45 seems to be missing in numbering only as text follows. UNIVERSITY MICROFILMS INTERNATIONAL 46 illuminations (0.5—3 seconds) with intervening dark periods to allow for the lag due to the relatively slow instrumental response time (t15 ; 0.5 seconds). The sum of the pH changes occurring as the result of each flash plotted against the total illumination time generated a reconstructed time-course of the pH-rise which could be used to calcu- late the rate of proton uptake. The kinetics of electron transport were measured in a similar manner in the same apparatus by substituting a Clark 02 electrode (t15 ; 2 seconds) for the pH electrode. Details of the technique are explained more fully in Appendix VI. The use of the flash-yield technique outlined above made trans- ient differences between initial rates and steady state rates quite ob- vious. Thus, the initial "pH gush" (t%.g_ 0.01 seconds) associated with the overall Hill reaction and heretofore attributed to the reduction of the plastoquinone pool (Izawa and Hind, 1967) was easily detected by this method (Appendix VI, Fig. 3). No corresponding initial fast phase in oxy- gen evolution was detected, however, a fact which throws some doubt on the involvement of the plastoquinone pool in the initial rapid pH change. No such initial rapid pH changes were associated with DBMIB re- duction, the rate of proton uptake (and 02 evolution) determined for the first flash being essentially the same as the rate determined for a subse- quent flash. Because the rate of proton uptake was practically linear for approximately the first 4 seconds of illumination when DBMIB was the elec- tron acceptor, and for about 3 to 4 seconds after the initial "pH gush" when methylviologen was the electron acceptor, it was possible to deter- mine accurately the initial rate of proton uptake from the reconstructed 47 time-course obtained by the flash-yield technique. Similarly, the rate of electron transport (as measured by changes in oxygen concentration) was completely linear with illumination time in both systems (Appendix VI, Figs. 3,4). The ratio of the rate of H+ uptake to the rate of electron transport (both measured by the flash-yield method) was taken to be an accurate reflection of the efficiency of proton accumulation in these sys— tems. The values determined for the H+/e' ratio in the H20 “PDBMIB reaction were relatively constant (0.35-0.51) over the entire pH range tested (6.2-8.15), averaging about 0.4. This low value is in contrast to H+/e' values _3 1.7 observed for the H20-—> MV reaction (Appendix VI, Table I, Fig. 4; see also Izawa and Hind, 1967). Thus the proton pump associated with DBMIB reduction (involving only coupling site II) is dis- tinguished from the proton pump associated with MV reduction in two ways: the reaction involving only site II lacks an initial rapid phase and is less than half as efficient as the combined sites in transporting H+. 4) Effect ofppH on the efficiencies of proton uptake (H+/e‘ and ATP formation_(P/e9)at coupling sites I and II. The remarkable simi- larity between the effect (or lack of effect) of pH on the H+/e' and P/e2 ratios associated with coupling site II prompted a further investiga- tion into the relationship between proton uptake and ATP formation at coupling site I. Figure 5 shows that the H+/e' values obtained for coupling site I only (using the Fe(CN)64'.e>MV reaction, see Izawa and Ort, 1974) were consistently around 1.0 regardless of the pH of the medium. The P/e2 48 men meowp_c:ou cowauommv A.¢mm_ .uso nae oZoNH :_ use HH> new H>._H mmOwncmqq< cw cm>wc .HH new H mmuwm m:w_gzou see mm:_o> In acmsmeewo um Ammpucwu EAFOmV m_mm;uc>m a~< new Ammpocwo cmgov mxopqz couosm do mmwu:m_u_emm ms“ eo comwcoaeou < .m weaned Ia w h m m N. m A « 611T... q _ ._ o lolBM/Iulollolllllloll 0\ I /olwo|.lo\o Tm o 0 I‘ll.\ H 35 0 TI 0/ O 0 II. o oo 1 H 35 _ _ _ _ _ L “l o ae/d Jo -a/,H 0.. 49 values obtained for the same reaction, however, show the marked pH de- pendence characteristic of site I phosphorylations (see Results section 81; see also Izawa and Ort, 1974). The reason for the lack of correla- tion between the efficiencies of proton uptake and phosphorylation at coupling Site I and the close correlation between these processes at coufling site IIiS not at all clear. The implications of this important difference between the two coupling sites will be dealt with more fully in the Discussion (see also Appendix VIII). 5) Effect of phosphorylation and arsenylation on proton pptake. Whether the proton gradient built up by Chloroplasts represents an oblig- atory intermediate of ATP formation, or an energy reservoir on a side- pathway, one might reasonably expect that energy utilization might lower the steady state level of the gradient (Mitchell, 1966). However, both inhibitions and stimulations of proton gradients by phosphorylation have been reported (Dilley and Shavit, 1968; McCarty etugl., 1971; Gould and Winget, 1972; Karlish and Avron, 1968). Because ATP formation can in- crease the rate of electron transport (and proton pumping) drastically, and because ions or ADP alone can greatly increase the extent of proton uptake (Dilley and Shavit, 1968; McCarty e3_§lg, 1971), the true effect of ATP formationron the proton gradient could easily be masked. However, use of the photosystem II dependent reduction of DBMIB avoids these com- plications since the electron transport in this system is not stimulated by phosphorylation (see Results, section 82) nor is proton uptake enhanced by the addition of ADP (Appendix VI, Table II). The effect of phosphorylation on the proton uptake in the ‘H20 —>DBMIB reaction was observed directly as: a) the extent of 50 dark-reversible H+ uptake superimposed upon the irreversible proton con- sumption due to ATP formation; b) the extent of the steady-state level of proton uptake in the presence of glucose plus hexokinose (to eliminate the irreversible proton consumption); and c) the extent of the steady- state level of proton uptake in the presence of arsenate instead of phos- phate (the unstable, arsenylated ADP hydrolyzes rapidly thereby elimini- nating the irreversible proton consumption). In each case a consistent lowering of the extent of proton uptake (40-60%) was observed when phos- phorylation (or arsenylation) occurred (Appendix VI, Table II, Figs. 5,6). DISCUSSION, DISCUSSION A. Functional Separation of the TWO Cogpling Sites in Chlorpplasts The results presented in this dissertation show clearly that it is possible to divide the chloroplast electron transport chain into two parts through the use of appropriate exogenous electron donors, electron acceptors and electron transport inhibitors. Each part uses only one of the two photosystems and each part uses only one of the two coupling sites. The photosystem II-dependent transport of electron from water to lipophilic oxidants such as p-phenylenediimines and p-benzoqui- nones (Class III acceptors) is coupled to phosphorylation only at coupling site II when the photosystem I-dependent component of the reduction is inhibited (Results, Section A; see also Appendicies I-IV). Similarly, the photosystem I-dependent transport of electrons from exogenous electron donors such as diaminodurene or reduced dichlorophenolindophenol to hydro- philic acceptors such as methylviologen or NADP+ is coupled to phosphory- lation only at coupling site I when the flow of electrons from photosystem II is inhibited (Results, Section B; see also Appendicies I-III, VII). These conclusions are supported by inhibitor studies. Thus the reduction of Class III acceptors is largely unaffected when the elec- tron transport chain is blocked at plastocyanin (Ouitrakul and Izawa, 1973), cytochrome f_(McCarty, 1974), or plastoquinone (Appendicies I-III). On the other hand, the reduction of Class III acceptors is extremely 52 53 sensitive to electron transport inhibitors which act Close to either the oxidizing side of photosystem II (Ort and Izawa, 1973) or the reducing side of photosystem II (Appendix II; Ouitrakul and Izawa, 1972). The oxidation of exogenous electron donors by photosystem I shows an entirely different pattern of responses to inhibitors; it is severely inhibited when electron transport is blocked at plastocyanin (Appendix I; Ouitrakul and IZawa, 1973) or cytochrome f_(McCarty, 1974), but it is completely in- sensitive to electron transport inhibition at plastoquinone (Appendix I), photosystem II or water oxidation (Appendicies I, III, VII). All of these findings are summarized in the scheme presented in figure 6 (see also Appendix VII). 8. Differences in the Properties of the Two CouplingpSites. The ability to separate and study the two coupling sites with- out resorting to disruption of the chloroplast lamellae by physical meth- ods or detergents made possible the discovery of a number of significant differences in the properties of the coupling reactions at sites I and II. These differences between the coupling sites are important in that they must be accomodated by any theory of the mechanism by which electron trans- port and ATP formation are coupled. 1) Regulation of electron flow py phosphorylation. One of the most obvious differences which can be observed experimentally is the lack of control of the rate of electron flow through site II by phosphoryla- tion. This lack of control can best be considered in the light of how electron flow is probably regulated by phosphorylation at site I. Portis e§_gl, (1975) have presented data which support the notion that an energy-dependent conformational change in CF1 actually .mgOpwn_::_ mzowsm> mo :o_uu< eo mmu_m mg“ new A i V cowuuznmcmcp xmcmcm mo mmpwm 03H mgu mcmzonm mummpacsopnu vmum—OmH cw mxngpma “sommcmsh cosuumpm so; msmgum < .m mszmwm Hmbm m wtm \lrx mxnwaL \11\ i... .1. __ .1. [Y __ ..| C 22 mar om EAT =8 l? , mallow: m p / aux oqmm mime :28 / e . //. 805800 / / F40 E $0.0 / .mqm 55 controls the rate of electron flow through coupling site I. In both the energized conformation and the deenergized conformation, CF1 is only slightly permeable to protons. However, according to these authors, when the energized conformation is being relaxed during phosphorylation, pro- ton permeability momentarily increases. This effectively lowers the "back pressure" of the internal prOtons on the proton producing electron transport reactions. However, it is possible that this back pressure represents an almost inconsequential amount of energy in the face of the overwhelming driving energy for the forward electron transport reaction supplied by photosystem II. At coupling site I, where the driving energy is considerably smaller, the back pressure could be considerably more sig- nificant, so that the rate of electron flow through this site would be a much stronger function of the proton gradient (see also Appendix VII), 2) HgCl2 inhibition. Izawa and Good (1969) first showed that mercurials inhibit coupled electron transport and ATP formation in a manner characteristic of energy transfer inhibitors. Energy transfer in- hibitors are believed to block phosphorylation by interfering with the terminal enzymatic steps of ATP synthesis, perhaps by binding to the coupling factor (CF1) or associated membrane proteins. Indeed the amount of HgCl2 required to attain the 50%inhibition plateau at coupling site I in chloroplasts (1 atom Hg++/40-50 chlorophyll molecules; Appendix VII) is in the same order of magnitude as the number of CF1 molecules associ- ated with the thylakoid (Murakami, 1968). It has been suggested that HgClz and other mercurials inhibit phosphorylation by binding to essential sulfhydryl residues (Izawa and Good, 1969; Bradeen and Winget, 1974) since HgCl2 inhibition is relieved 56 by cysteine but not by other chelators of Hg++. Further evidence that a sulfhydryl may be involved comes from the unusual effect of HgCl2 on electron transport in the absence of both ADP and Pi“ Under these condi- tions the rate of electron transport is stimulated by HgCl2 to the level of coupled electron flow in the absence of HgClZ(Izawa and Good, 1969). The only other energy transfer inhibitor reported which exhibits this property is N-ethylmaleimide, which reacts with a sulfhydryl residue on the y-subunit of CF1 (McCarty et_gl,, 1972; McCarty and Fagan, 1973). Bradeen et_gl,, (1973) concluded that HgCl2 probably did not inhibit CF1 directly Since the formation of ATP in the dark after an il- lumination (XE) was much less sensitive to the inhibitor than steady-state phosphorylation. However, this insensitivity has been observed with other energy transfer inhibitors as well, and probably reflects the introduction of different rate-limiting steps in XE phosphorylation. In any event, it seems likely that mercurials, like other energy transfer inhibitors, inter- fere with phosphorylation at a point close to the terminal ATP synthes- izing process. If it is true that mercurials serve as energy transfer inhibi- tors by reacting with the coupling factor, the very great difference in the sensitivities of site I and site II is of paramount importance; here- tofore there has never been any evidence that different coupling sites use different coupling factors, and the chemiosmotic hypothesis specifies a common coupling factor. Finally, it is interesting to note that the lipophilic mercurial p-hydroxmercuribenzoate is much less site-specific than HgClz, raising the interesting possibility that the mercury sensitive component may be in 57 different microenvironments at the different coupling sites. This pos- sibility will be discussed in more detail below. 3) The effects ofng on the efficiences of phosphorylation (P/e2)_ and_proton uptake (Hf/e7). It is not at all difficult to understand that the efficiency of proton uptake associated with electron transport through each coupling site is insensitive to the pH of the medium. There is good reason to believe that proton uptake is a consequence of antisotropic arrangement of the electron carriers and hydrogen (i.e. H+ plus e”) carriers in the membrane. The reduction by an electron of a hydrogen carrier near the outer side of the membrane results in the uptake of a proton from the medium, and the oxidation of the hydrogen carrier by an electron carrier near the inner side of the membrane results in the loss of a proton to the inside of the thylakoid. In the case of coupling site II, the oxidation of H20 at the inner membrane interface is almost certainly the source of the internal protons. The ratio of protons trans- located to electrons transported can only be affected by pH if i) the apparent hydrogen carrier has a pka within the pH range being investigated, loses a proton, and is therefore not really a hydrogen carrier at all, or ii) changing the medium pH drastically alters the arrangement of the membrane so that the release of protons by hydrogen carrier upon oxida- tion is no longer toward the inside of the thylakoid. The fact that changing the medium pH causes no significant change in the H+/e' ratios suggests that neither of these possibilities is occurring over the pH range 6-9 (Appendix VI, VIII). Even though changing the pH of the medium has no effect on the ef- ficiency of H+ uptake, changing the pH does affect the efficiency of site 58 I-dependent phosphorylation dramatically. In contrast, the efficiency of site II phosphorylation is hardly affected. Below pH 7, the efficiency of ATP formation supported by site I has fallen to nearly zero, while the efficiency of ATP formation supported by site II is practically unchanged. What this seems to say is that at low external pH, whether or not ATP is made depends upon exactly where (i.e. at which electron transport reac- tion) the internal protons are generated. Again, as in the case of Hg++ inhibition, low pH seems to be a site-specific inhibition of phosphoryla- tion, which cannot be easily accomodated by the chemiosmotic hypothesis. C. The Cpupling Mechanism and Coupling Site-Specificity Attempting to explain the data described above within the framework of existing hypotheses of energy conservation is an illuminating exercise since,it forces one to define more precisely the molecular mechanism in- volved. The evidence which has accumulated in support of some sort of Chemiosmotic coupling mechanism (as detailed by Mitchell, 1966) makes the chemiosmotic hypothesis attractive. The site-specificities exhibited by Hg++ and pH can be fitted into the framework of the chemiosmotic hypoth- esis as outlined below. Williams (1969) has suggested that the actual pH gradient involved in energy coupling is confined to a highly localized area of the membrane, and is in equilibrium with a delocalized, transmembrane gradient. Accept- ing this postulate, one can construct a model for energy coupling which allows site-specificity while at the same time preserving most of the fundamental principles of the chemiosmotic hypothesis. According to the model, the localized pH gradient within the membrane is in the vicinity of a CF1 molecule. The CF1 molecule utilizes the protons in the 59 intramembrane gradient to drive phosphorylation, while any extra protons which are generated by the electron transport reactions are released to the inside of the thylakoid to establish the transmembrane gradient. Thus, protons produced at one coupling site would not be available to support ATP formation at another coupling site except via the relatively slow equilibration of the localized gradients via the transmembrane gradient. The site-specific effects of Hg++ and pH can then be understood by postu- lating that that portion of the energy conserving appratus associated with coupling site II which is sensitive to Hg++ and low pH is in a more hydrophobic environment than the corresponding portion at the apparatus associated with coupling site I and therefore is less accessible to mer- curic or hydrogen ions. There is some evidence which supports such a model. For example, it is very likely that the water-splitting reaction, the H+ generator for site II, is located within a hydrophobic region of the membrane. Further- more, 1ipophilic mercurials (e.g. p-hydroxymercuribenzoate) are much less site specific than mercuric Chloride (Gould, 1975b). This is consis- tent with the idea that the inhibition sites are indeed in different micro- environments. However, antiserum against CF1 shows no site specificity, an observation which indicates that, if there are different coupling factors associated with different coupling sites, at least a part of the CF1 mole- cule at each coupling site must be exposed to the aqueous environment. Of course, the observation does not preclude the possibility that some other essential portion of the coupling apparatus besides CF1 is buried in a hydrophobic environment. 60 It should be understood that the modification of the chemiosmotic hypothesis proposed above does not necessarily require that the coupling factor itself be directly associated with the coupling sites and in dif- ferent environments. One can equally well postulate that the suggested local accumulation of hydrogen ions interacts with CF1 through additional transducers which are unique to each coupling site. 'Still other models which could accomodate the apparent site- specificities of Hg++ and low pH might be possible, and given enough in- genuity it might be possible to make some of these models conform more closely to the chemiosmotic hypothesis as defined by Mitchell (1966). Final resolution of this matter must await evidence from new and different experiments utilizing new and different approaches. LITERATURE CITED Arnon, 0. Avron, M. Avron, M. BOhme, H. BOhme, H. BOhme, H. Bradeen, Bradeen, Brand, J. Brand, J. LITERATURE CITED 1. 1949. Copper enzymes in isolated chloroplasts. Poly- phenoloxidase in Beta Vulgaris. Plant Physiol. 24: 1-15. and B. Chance. 1966. Relation of phosphorylation to electron transport in isolated chloroplasts. Brookhaven Symp. Biol. 19: 149-160. and J. Neumann. 1968. Photophosphorylation in Chloroplasts. Ann. Rev. Plant Physiol. 19: 137-166. , S. Reimer and A Trebst. 1971. The effect of dibromothy- moquinone, an antagonist of plastoquinone, on non-cyclic and cyclic electron transport. Z. Naturforsch. 266: 341-352. and W.A. Cramer. 1971. Plastoquinone mediates electron trans- port between cytochrome b559 and cytochrome f_in spinach chloroplasts. FEBS Letts. 15: 349-351. and W.A. Cramer. 1972. Localization of a site of energy coupling between plastoquinone and cytochrome f_in the electron transport chain of spinach chloroplasts. 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Phosphorylation coupled to the oxidation of ferricytochrome c. J. Biol. Chem. 215: 555-570. Oliver, D.J. and A.T.Jagendorf. 1975. Inhibition of the coupling factor from spinach chloroplasts by trinitrobenzenesulfonic acid. Fed. Proc. 34: 596. Ort, D.R. and S. Izawa. 1973. Studies on the energy coupling sites of photophosphorylation. II. Treatment of chloroplasts with NH,OH plus EDTA to inhibit water oxidation while maintaining enérgy coupling efficiencies. Plant Physiol. 52: 595-600. Ort, D.R., S. Izawa, N.E. Good and D.W. Krogmann. Effects of the plasto- cyanin antagonists KCN and poly-L-lysine on partial reactions in isolated chloroplasts. FEBS Letts. 31: 119-122. Ouitrakul, R. and S. Izawa. 1973. Electron transport and photophos- phorylation in Chloroplasts as a function of the electron ac- ceptor. II. Acceptor-specific inhibition by KCN. Biochim. Biophys. Acta 305: 105-118. Polya, G.M. and A.T. Jagendorf. 1969. Light-induced change in the buffer capacity of spinach chloroplast suspensions. Biochem. Biophys. Res. Commun. 36: 696-703. Portis, A.R., R.P. Magnusson and R.E. McCarty. 1975. Conformational changes in coupling factor 1 may control the rate of electron flow in spinach chloroplasts. Biochem. Biophys. Commun. 64: 877-884. Racker, E. and W. Stoeckenius. 1974. Reconstitution of purple membrane vesicles catalyzing light-driven proton uptake and adenosine triphosphate formation. J. Biol. Chem. 249: 662-663. Ryrie, I.J. and A.T. Jagendorf. 1971. An energy-linked conformational change in the coupling factor protein in chloroplasts. Studies with hydrogen exchange. J. Biol. Chem. 246: 3771-3774. 67 Ryrie, I.J. and A. Jagendorf. 1972. Correlation between a conformation- al change in the coupling factor protein and the high energy state in chloroplasts. J. Biol. Chem. 247: 4453-4459. Saha, S. and N.E. Good. 1970. Products of the photophosphorylation reaction. J. Biol. Chem. 245: 5017-5021. Saha, S., R. Ouitrakul, S. Izawa and N.E. Good. 1971. Electron trans- port and photophosphorylation in chloroplasts as a function of the electron acceptor. J. Biol. Chem. 246: 3204-3209. Siggel, U., G. Renger, H.H. Stiehl and B. Rumberg. 1972. Evidence for electronic and ionic interaction between electron trans- port chains in chloroplasts. Biochim. Biophys. Acta 256: 328-335. Slater, E.C. 1953. Mechanism of phosphorylation in the respiratory chain. Nature 172: 973- 978. Stiehl, H.H. and H.T. Witt. 1969. Quantitative treatment of the function of plastoquinone in photosynthesis. Z. Naturforsch. 24b: 1588-1598. Trebst, ., E. Harth and W. Draber. 1970. On a new inhibitor of photosynthetic electron-transport in isolated chloroplasts. Z. Naturforsch. 25b: 1157-1159. Williams, R.J.P. 1969. Electron transfer and energy conservation. In Current Topics in Bioenergetics 3, D. R. Sanadi, ed. ,Academic_ Press, New York. pp. 79-156. Winget, 6.0., S. Izawa and N.E. Good. 1966. The stoichiometry of photophosphorylation. Biochem. Biophys. Res. Commun. 21: 438-443. Winget, 6.0., S. Izawa and N.E. Good. 1969. The inhibition of photo- phosphorylation by phlorizin and closely related compounds. Biochemistry 8: 2067-2074. APPENDICES APPENDICES The following appendices are included as reference material for a large number of experimental data. Appendix VIII represents a summary of much of this work, along with a more complete discussion of the pro- posed modifications of the chemiosmotic hypothesis. The preponderant portion of the data in the appendices are data obtained in the course of the work described in this dissertation. However, some of the data in some of the appendices represent the work of others. It is impossible to assess the relative contribution of this author to each of these works because of the high degree of interaction and collaboration among this author and his colleagues during the course of these investigations. 69 APPENDIX I ELECTRON TRANSPORT AND PHOTOPHOSPHORYLATION IN CHLOROPLASTS AS A FUNCTION OF THE ELECTRON ACCEPTOR III. A DIBROMOTHYMOQUINONE- INSENSITIVE PHOSPHORYLATION REACTION ASSOCIATED WITH PHOTOSYSTEM II 71 Reprinted from Biochimica et Biophysica Acta, 305 (I973) 1 19-128 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 46544 ELECTRON TRANSPORT AND PHOTOPHOSPHORYLATION IN CHLORO- PLASTS AS A FUNCTION OF THE ELECTRON ACCEPTOR III. A DIBROMOTHYMOQUINONE—INSENSITIVE PHOSPHORYLATION REACTION ASSOCIATED WITH PHOTOSYSTEM II' S. IZAWA. .1. MICHAEL GOULD, DONALD ‘R. ORT, P. FELKER and N. E. GOOD Department of Botany and Plant Pathology, Michigan State University, East Lansing, IWicli. 48823 (U.S./l.) (Received January 8th, 1973) SUMMARY Dibromothymoquinone (2.5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) is reputed to be a plastoquinone antagonist which prevents the photoreduction of hydrophilic oxidants such as ferredoxin—NADPT. However, we have found that dibromothymoquinone inhibits only a small part of the photoreduction of lipo- philic oxidants such as oxidized p-phenylenediamine. Dibromothymoquinone-resistant photoreduction reactions are coupled to phosphorylation. about 0.4. molecules of ATP consistently being formed for every pair of electrons transported. Dibromo- thymoquinone itself is a lipophilic oxidant which can be photoreduccd by chloro- plasts. then reoxidized by ferricyanide or oxygen. The electron transport thus catalysed also supports phosphorylation and the P/e2 ratio is again 0.4. It ?s concluded that there is a site of phosphorylation before the dibromothymoquinone block and another site of phosphorylation after the block. The former site must be associated with electron transfer reactions near Photosystem 11, while the latter site is presum- ably associated with the transfer of electrons from plastoquinone to cytochromef. INTRODUCTION Two quite different arguments lead us to the conclusion that non-cyclic photo- phosphorylation involves more than one site of energy conservation. Our reasons for believing that there are at least two phosphorylation sites are as follows: (I) The overall efi‘iciency of photophosphorylation (P/ez) is considerably higher than one ATP molecule formed for every pair of electrons transportcd'. Furthermore, the We, ratio approaches 2.0 if one subtracts that part of the electron transport which can occur in the absence of phosphorylation’. (2) Lipophilic strong oxidants (Class III acceptors). such as the oxidized form of p-phenylenediamine, intercept electrons by reacting with some intermediate Abbreviations: DCMU, 3-(3,4-dichloropheny!)-l,l-dimethylurea; P/ec. ratio of the mole- cules of ATP formed to the pairs of electrons irarmoried. ' Journal Article No. 6219 of the Michigan Agricultural Experiment Station. 72 120 S. IZAWA ('1 0]. carrier which normally transfers electrons from Photosystem II to Photosystem I (refs 3, 4). This interception of electrons does not abolish phosphorylation but instead decreases the elliciency to about half of the value observed when hydrophilic Class I acceptors are reduced. It therefore seems that the intermediate carrier re- sponsible for the reduction of oxidized p-phenylenediamine is situated between two sites of phosphorylation in the electron transport chain. The work described in this paper was undertaken in an attempt to define the location of the two phosphorylation sites in terms of the sites of action of known electron carriers. It has long been thought that there must be a rate-determining, phosphorylation~ dependent reaction transferring electrons between the two pltotosystems5’7. This rate-determining step presumably lies between plastoquinone and cytochrome f since the rate of reduction ofcytochrome/by Photosystem ll and the rate ofoxidation of plastoquinone by Photosystem l are accelerated during phosphorylation or when uncouplers are added. However, there is good reason for doubting that this rate- determining phosphorylating process is involved in the reduction of oxidized p- phenylenediamine. The reduction of oxidized p-phenylenediamine is very fast and independent of phosphorylation’. Moreover, the fact that 3-(3,4-dichlorophenyl)- l,l-dimethylurea (DCMU) inhibition of oxidized p-phenylenediamine reduction is independent of light intensity suggests that oxidized p-phenylenediamine accepts electrons from a carrier situated close to Photosystem II“. We wish now to present evidence which lends further support to the concept of a site of phosphorylation close to Photosystem II and at the same time virtually precludes the participation of a plastoquinone—cytochrome f phosphorylating re- action in oxidized p-phenylenediamine reduction. The new evidence has been provided by studies of the effects ofdibromothymoquinone(2,5-dibromo-3vmetliyl-6-isopropyl- p—benzoquinone) on electron transport and phosphorylation. This inhibitor was lirst introduced by Trebst and his associates” as a plastoquinone antagonist. At very low concentrations it blocks all ofthe transfer of electrons from water to Class I acceptors such as ferredoxin—NADP+ or methylviologen and a large part of the transfer of electrons from water to ferricyanide". It also seems to block the transfer of electrons from cytochrome bssg to cytochrome f and it certainly prevents the reduction of cytochrome/by electrons from Photosystem Il'°"'. These observations do indeed suggest that the inhibitor acts at the level of plastoquinone involvement. In any event,it is clear that the inhibitor prevents electron transport at some point after Photosystem II but before cytochrome f. Yet dibromothymoquinone does not greatly inhibit either oxidized p-phenylenediamine reduction or the associated phosphorylation reaction. It follows that there must be a site of phosphorylation before the site of dibromothymoquinone inhibition and probably therefore before the site of involvement of plastoquinone. MATERIALS AND METHODS The procedures employed in this study were similar to those employed in the earlier papers of the series’". Chloroplasts were isolated from commercial spinach (Spi‘naeia oleracea L.) as already describt-J’. Cyanide-treated chloroplasts were pre- pared by incubating chloroplasts at 0 ’C for 90 min in a 30 mM KCN solution 73 PHOTOSYSTEM II PHOSPHORYLATION lll buffered at pH 7.8 as described in the previous paper‘. Control chloroplasts were suspended for the same time in a similar medium containing KOH instead of KCN. The inhibitor dibromothymoquinone was prepared by bromination of thymo- quinone in water and was recrystallized several times from alcohol. Stock solutions were prepared by dissolving dibromothymoquinone in ethanol—ethylene glycol (1:1, v/v). The concentration of the stock was such that the organic solvent in the reaction mixture never exceeded 1%. The reduction of ferricyanide was measured as the decrease in absorbance of the reaction mixture at 420 nm. In experiments with oxidized p-phenylenediamine. recrystallized colorless p-phenylenediamine dihydrochloride was added to the bulfered reaction mixture, then oxidized immediately before the reaction with excess ferri- cyanide. Electron transport was measured as reduction ofthe excess ferricyanide since the oxidized p-phenylenediamine reduced during the reaction is immediately re- oxidized by ferricyanide. Reactions involving other aromatic diamines and quinones were measured in the same indirect way. The reduction of methylviologen was measured as oxygen uptake since reduced methylviologen reacts rapidly with oxygen to form H202”. For these measurements a Clark-type. membrane-covered electrode was used. Phosphorylation was measured by a modification ofthe method of Avron' 3 as the residual radioactivity after extraction of the 33P-labeled orthophosphate from the reaction mixture as phosphomolybdic acid. In all experiments the tempera- ture was 19 "C. RESULTS (I) The sensitivity of electron transport and phosphorylation to (lt'ln-mnot/tt'mnqmnone wit/i cltflerent electron acceptors As we have reported elsewhere’, lipophilic oxidants tend to increase the rate of electron transport in illuminated chloroplasts. decrease the dependence of electron transport on phosphorylation and reduce the eliiciency of phosphorylation (Pg’ez) toward one-half. The extent to which acceptors are able to intercept electrons between two phosphorylation sites can be roughlyjudged by the increase in the rate ofelectron transport and the decline in the Pm: ratios. (These criteria only apply. of course, if the We; ratios fall to a plateau rather than to zero and it can be shown that the acceptor is not an uncoupler.) Among the lipophilic acceptors listed in Table oxidized p-phenylenediamine is most nearly a typical Class Ill acceptor while 2 dimethyl-p-benzoquinone is the least typical. Ferricyanide ion is not lipophilic at all and. in our chloroplasts. seems to intercept very few of the electrons generated by Photosystem ll. As can be seen in Table l, the transport ol'electrons to lipophilic acceptors has a large component which is resistant to dibromotliymoquinone. This component is largest with the best Class III acceptor, oxidized p-phenylenediamine and smallest with the worst Class III acceptor. 2.6-dimethyl-p-benzoouinone. Clearly. that part of the electron transport which results from the interception of electrons between the phosphorylation sites is largely insensitive to the inhibitor. This is even more obvious when one notes the ell’ect of dibromothymoquinone on the Pje2 ratios. Regardless of the ratio in the absence of the inhibitor. that is regardless of what proportion of the electrons are intercepted between the phosphorylation sites. the P/e2 ratio always falls to about 0.4 in the presence of the inhibitor. This is true even I. .5- 74 '22 S. IZ--\\V.I\ et al. TABLE I THE EFFECT OF DIBROMOTHYNIOOUINONE O\l ELECTRON TRANSPORT AND i’HOTOl’l-IOSPHORYLA'I‘lON IN CHLOROPLASTS WITH DIFFERENT ELECTRON ACCEPTORS The 2.0-ml reaction mixture consisted of the following: 0.l M sucrose. 50 mM Tricine buffer (pH 8.2), 2 m.\1 MgCl-g, 1 mM ADP, 5 mM 3'3l’:. chlorOplasts containing 30/Ig chlorophyll, and the indicated acceptor system. These acceptor systems were: 0.5 m.\l potassium ferricyanide (Fecy); 0.5 mM p-phenylenediamine plus l.5 mM ferricyanide (PDM); 0.5 mM diaminodurene plus l.5 mM ferricyanide (DADux): 0.5 mM 2.5-dimetii;.'l-p~benzoqtiinone plus 0.5 mM ferri- cyanide (DMQ): 0.5 mM 2,5-diaminotoluene plus 1.5 mM ferricyanide (DATq). When used dibromothymoquinone was 0.5/1M. Rates are expressed in Ineqtiiv or ,lsnoles ATP h per mg chlorophyll. Electron Rate of electron transport Rate of ATP/bruiatinn P, e: acceptor _ ‘ _ “ . Control + dibromu- Control + ill/'irnmo- Control + (ll/V‘UHIO- t/ti'mm/nlnone t/tymnt/m't'otie t/ti-moqninone Fecy 430 SS 228 I 3 l.0ti 0.45 PD.“ 1260 695 293 149 0.46 0.43 DAan 735 383 244 50 0.66 0.39 DMQ .902 294 325 52 0.72 0.36 DATux 791 390 280 95 0.71 0.48 for the tiny residue of electron transport with ferricyanide as acceptor. We have also found that, regardless ofthe acceptor, the dibromothymoquinone—resistant component of the electron transport is always independent of the presence or absence of ADP and phosphate. Further elTects of dibromothymoquinone are illustrated in Figs l—4. Again. the transport of electrons from water to oxidized poplienylenediamine has two components: one large, insensitive to dibromothymoquinone and supporting phos- phorylation with a P/e2 ratio of about 0.4: the other smaller. sensitive to dibromo- thymoquinone with a computed P’ez about 1.0 (Fig. I). In contrast. the transport of electrons to ferricyanide is mostly sensitive to the inhibitor while the transport to methylviologen is almost all sensitive (Fig. 2). Once again the small residue of dibromothymoquinone-insensitive ferricyanide reduction supports phosphorylation with a P/el ratio of 0.4-05. This residual dibromothymoquinone-resistant ferricyanide reduction deserves attention since we have here the unusual situation of an inhibitor seeming to catalyze the reaction it inhibits; increasing concentrations of dibromothymoquinone actually increase the rate of ferricyanide reduction (Figs 3 and 4). The inhibitor, in addition to blocking electron transport. is itself a lipophilic oxidant which accepts electrons before or at its own site of inhibition. Apparently the reduced dibromothymoquinone is quickly reoxidized by ferricyanide and thus the inhibited ferricyanide reduction is in part restored. However, this dibromothymoquinone-mediated ferricyanide reduction is quite dilTerent from the usual ferricyanide Hill reaction. having instead many of the characteristics of oxidized p-phenylenediamine reduction: the rate is independent of the presence or absence of .\DP and phosphate or of uncouplers such as methylamine. and the elliciency of phosphorylation (l’fez) is only 0.3 4).-l. .-\l:.‘ioagh PHOTOSYSTEM II PHOSPHORYLATION 123 o o 2 io’Ml I W 1 - u ' . l 7' l , l 7 r MV ‘\___.___1___.. E 7‘8— \tv 0" - 0' ibi- : = + l Fecy -‘ l B 1 i \ 500 -1' a. l\ a“ : ..\ i . L :1 a 0.5». \o- XX are (900.) E _i‘_\\ l " 33°. ““““ _ :3 l E}; z !\\\ J I 11 l \ K" (X 00 f ' 250 A ATPU’OCV) E Deromotnymoquiflonexto 'M) “ z _i_ \t‘ _l \ EItFt-cy) g ‘ ‘ ° up ' - ' er. A 0‘ _ i w l 9: l . x—e : . . . .\,.- ‘ A I _11 i _ 00 l 2 3 4 5 00 l 2 ” s o l fi’t‘?‘ Dibmmothymominone x107(M) Dibromothymoquinone x 107 (M) Fig. 1. Effect of dibromothymoquinone on electron transport (E.T.) and phosphorylation (ATP) with ferricyanide (Fecy) or oxidized p-phenylcnediamine (9130:) as electron acceptor. Rates are expressed in nequiv or pmoles ATP/h per mg chlorophyll. The 2. O-ml reaction mixture consisted of the following: sucrose. 0.] M; Tricine NaOH buli't r (pll 8 .2). SO mVl; MgClz. 2 mM; ADP, 1 mM; 3- -P:, 10 mM; chloroplasts containing 40”: (fern. ' - t '-.\ or Song (3 an) chlorophyll; and either 0.5 mM potassium ferricyanide or a combination 1.5 mM ferricyanide and 0.5 mM p-phenylenediamine dihydrochloride. Note the great sensitivity of ferricyanide reduction to the inhibitor, the relative insensitivity of oxidized p-phenylenediamine reduction and the high rate of ATP formation associated with the resistant oxidized p-phenylencdiamine reduction. Fig.2. Effect of dibromothymoquinone on electron transport and phosphorylation with ferri- cyanide and methylviologen (MV) as electron acceptors. Reaction conditions, units and ab- breviations as in Fig. 1 except that 32P: was 5 mM, potassium ferricyanide was 0.4 mM and methylviologen was 50 pM. Electron transport was measured as oxygen produetion with ferri- cyanide and as oxygen consumption with methylviologen. The dotted curve for oxidized p- phenylenediamine (PD...) in the inset figure is taken from Fig. l for comparison. Note from the inset figure that the small amount of residual ferricyanide reduction supports phosphorylation with the efficiency characteristic of the oxidized p-phenylenediamine-reducing system. Presumably ferricyanide can intercept electrons, either directly or indirectly, between two sites of phospho- rylation as can oxidized p-phenylenediamine whereas methylviologen cannot. this catalysis of ferricyanide reduction is most conspicuous at high dibromothymo- quinone concentrations, there is no reason to doubt that it is already taking place at the point of apparent maximum inhibition of ferricyanide reduction and even there constitutes a large fraction of the residual reaction. This is clearly indicated by the fact that the We; ratio associated with ferricyanide reduction declines to 0.4-0.5 as the dibromothymoquinone inhibition approaches its maximum (Figs l and 2). No such decline is observed when the low potential acceptor methylviologen is being reduced; for thermodynamic reasons reduced dibromothymoquinone cannot donate electrons to methylviologen and therefore the inhibitor cannot catalyze methyl- viologen reduction. 76 l24 S. IZAWA at u/ // . -'-—"7"‘ “"_‘ f I /.« T i v “'“r‘”! ‘— a - ' . ' . l I ' ' / t‘} south '1, 300‘.- l —— ' '3 ‘ 1.0g l Q J . Ali. : i ‘ Fecy ’ z ‘i’ l a ' .l . ‘. *- ‘ 4" l ' VI \ i (I I ., ‘05 'l. 5 .3“ its , e “L 1’3. 1 \ ~ I 3~°‘—-—0 - {v . e 0-5 :3 _j o . ° l _ -.- i \ , _ A 20C - $200 I , o—o-- , o ‘- :SJ 01 1 I 1 t J 1 g... .1 l ,. \ o 2 a e s _ l'.‘ . g ;‘ O L——L 1 1 L L i : ! Dibromotnyrrmyuinonc i. '7" z ,2", l a l: O 2 a 6 8 V) m i ' u) if. Dibromothymo :uinonex 106 (M) ‘ 0 ll' 1 <21 ii -O'i 23 iii E I (Fecy) i l -, ‘. . ' O-—-—-_ :2 i. o/ .3 a: \/o/'__ -—o-. 2 i ' '- i I Q l i .7 i ‘- \O/ - LI ii ‘/ f “i, ii & i- ;i O\ /(J Artur-9c,” ‘ 2 Al‘ ATP‘lFCCV) i Q \o/ ‘, _...z r- .) \ 7‘ ._..—-—-0 __ . \. //". F r (W) ATPlMVl1 w ii I it (W, are Ca in i‘ ‘1_____3___.£_‘ _____.‘ ’ j l 0:2—-—--~—- ~$'——-—-/wvi l O Lmr:‘ :‘""M+—/,// O . HTT“I"’" "“"T'T—T' J c ; 2 3 4 10 o : 2 3 a " IO Dibromotr'iymociuinonex10‘5 (M) Dibmmothymoouinone MD6 (M) Fig. 3. Ell‘ect of higher concentrations of dibromothymoquinone on electron transport and phos- phorylation with ferricyanide or methylviologen as acceptors. Reaction conditions, units and abbreviations as in Fig. 2. except that methylviologen was lOOnM. Note the much greater residual electron transport with ferricyanide and the increasing rate of ferricyanide reduction with in- creasing dibromothymoquinone concentration. Note also from the inset ligure that the inhibitor- insensitive electron transport again supports phosphorylation with the elliciency characteristic of the oxidized p-phenylenediaminc-reducing system. Apparently the inhibitory lipid soluble quinone dibromothymoquinone is reduced by chloroplasts, in a reaction \vhicn does not include the site ofdibromothymoquinone inhibition and is then rapidly reoxidized by the excess ferricyanide. Fig. 4. Echcts of dibromothymoquinone on digitonin-trcated chloroplasts. Conditions. units and abbreviations as in previous figures. Chloroplasts were. treated with 0.”.‘".', digitonin in bull'er (pH 7.4) at O ' C for 15 min. spun down at 4000xg for 10 min and washed twice in butler. The chloroplast material subjected to this mild treatment consisted in the main part of untragmented lamellae or large fragments. Rates of electron transport and phosphoryiation vvere only slightly lowered by the treatment. Note. however, that the treatment lowered the concentration of dibromothymoquinone required to cataly/e ferricyanide reduction (see also fig. 3). thereby pro- ducing the illusion that ferricyanide reduction is in large par: insensitive to the inhibitor. The dibromothymoquinone reduced by chloroplasts can react with oxygen to produce H303 when ferricyanide is not present. his dibroniothymoqainone- insensitive Mehler reaction becomes substantial when the concentration of inhibitor is above 10 /iM. As will be described in another paper. this reaction also supports phosphorylation with a P/ez ratio of 0.4. Thus the reaction provides : mechanism for "pseudocyclic" photophosphorylation which probably involves only Photo- system ll. A similar reaction -— the reduction of dibromothymoquinone by illuminated chloroplasts and its subsequent reoxidation by oxygen in the dark" has been noted by Lozier and Butler”. All of the dibromothymoquinone-resistant reactions we have tested, electron transport and phosphorylation alike. are inhibited by DCMU which inactivates Photosystem II and are largely insensitive to KCN which inactivates plastocyanin. 77 l’llOTOSYSTEM ll PHOSPHORYLATION lIS The observations described above concerning the effect of dibromothymo- quinone or ferricyanide are somewhat at variance with the observations of Zohme er al.°. They found that nearly half of the transport of electrons to ferricyanide in “intact" chloroplasts was resistant to dibromothymoquinone. while in our chloro- plasts the resistant portion never exceeded 20".. and was sometimes much less. Presumably this discrepancy resulted from some dill’crence in the state of the chloro- plast membranes. As shown in Fig. 4 (compare with Fig. 3). the ferricyanide reduction became markedly less sensitive to dibromothymoqainone when the membranes were slightly modified by treating the chloroplasts with a low concentration of digitonin. A more serious discrepancy. however. is to be found in the fact that the dibromo- thymoquinone-resistant ferricyanide reduction in our chloropiasts remains lirmly coupled to phosphorylation. A constant PM: ratio ol'().3~—().4 is observed over a wide range of conditions. even alter the digitonin treatment. ln contrast. the data of BOhme er 0!. show a continual decline in phosphorylation elliciency with increasi g dibromothymoquinone until a Pie2 ratio of zero is reached (see Fig. 2 of ref. ll. Bohme at (1]. concluded from their observations that the dibromothymoauint ie- insensitive portion of ferricyanide reduction is not coupled to phosphorylation where- as we are forced to conclude from our observations that it is coupled. The cause of the discrepancy is as yet unknown. We agree with Bt'ihme er u/. that the reduction of ferricyanide by sonicated chloroplasts is quite resistant to dibromothymotiumone. but here again we noted that considerable phosphorylation was associated with the dibromothymoquinone-insensitive e.ectron transport. In a sonicated chloroplast preparation the P’ez ratio was 0.2 with inhibitor and 0.6 without inhibitor. Presum- ably further disruption would have still further increased the resistance to the iii- hibitor while abolishing phosphorylation in dibromothymociuinone-treated and control alike. (2) E videncc bearing (m the .vi'lc of dibromnH'ii'moqiri'iimic inhibition (T able ll ) Our observation that there is a site of phosphorylation on the electron trans- port pathway before the dibromothymoquinonc inhibition site makes precise identiti- cation of the inhibition site a matter of critical concern. There is already evidence that dibromothymoquinone interferes with electron transport at the level of p‘asto- quinone"‘ll but this evidence is not absolutely conclusive. Moreover. the most striking features of dibromothymoquinone inhibition described here (the strong inhibition ofelcctron transport with hydrophiiic acceptors. the weak inhibition with lipophilic acceptors. and the lowering of the Pie: ratio to 0.41 are also characteristic of KCN inhibition‘. Yet KCN almost certainly inhibits because it reacts with plasto- cyanin. Therefore. it seemed important to us to prove that the site of dibromothymo- quinone inhibition is different from and precedes the site of KCN inhibition. Table ll shows that the elfects ol' the two inhibitors are indeed quite distinct. DCMU-insensitive, Photosystem l-depcndcnt reactions such as the transport of electrons from diaminodurene to methylviologen and the diazninodureiie-iiiediated cyclic phosphorylation system are inhibited by KCN but. as llohine ('l ul." have already implied, are not inhibited by dibromothyinoqtiinone. The transfer ol'electrons from water to Class I acceptors such as methylviologen is inhibited by both KCN and dibromothymoquinonc. in contrast. the [DC‘vIUsensitiv'e transfer of electrons from water to oxidized p-pheiiylenediaininc is inhibited by neither. This we must 78 126 ’ S. IZAWA er al. TABLE II INHIBITION OF VARIOUS REACTIONS IN CHLOROPLASTS BY DIBROMOTHYMO- QUINONE AND KCN Reaction conditions and concentrations of reactants were as in Table I and the figures unless otherwise specified. Units are as in Table I. PDox represents products of the oxidation of p- phenylenediamine. When KCN was used, the chloroplasts were pretreated as described in Materials and Methods. When dibromothymoquinone was used, it was 0.5 i" M. Density of chloro- plast suspended in the 2.0 ml reaction mixture was: water—i» methylviologen, 40 pg chlorophyll; water» PDox, 30/ig chlorophyll; diaminodurene» methylviologen and diaminodurene (cyclic), lOpg chlorophyll. In the diaminodurene»methylviologen system ascorbate (l mM), DCMU (liiM), diaminodurene (0.5 mM) and methylviologen (0.1 mM) were added and the pH was lowered to 7.7 to eliminate much of the non-biological ascorbate oxidation. The diaminodurene (cyclic) phosphorylation system was similar except that methylviologen and ascorbate were omitted and 0.1 mM ferricyanide was added to establish an appropriate diaminodurene,’oxidized diaminodurene ratio. System Condition Electron A 77’ P/e 2 transport formation Water—r methylviologen Control 646 395 1.13 +dibromothymoquinone 44 6 — KCN treated 0 0 —— Water» PDox Control 1720 386 0.45 + dibromochymoquinone 1300 257 0.40 KCN treated 1200 198 0.33 Diaminodurene—r methylviologen Control 4130 733 0.35 +dibromothymoquinone 4580 705 0.31 KCN treated 440 33 (0.15) Diaminodurene (cyclic) Control —- 702 — 4-dibromothymoquinone — 605 — KCN treated — 12 —- conclude that the site of dibromothymoquinone inhibition falls between the DCMU inhibition site and the KCN inhibition site. This is consistent with the view that di- bromothymoquinone acts as a plastoquinone antagonist. .DISCUSSION In the first paper of this series3 we noted that lipophilic strong oxidants (e.g. oxidized p-phenylenediamine) can be reduced very rapidly by illuminated chloro- plasts whether or not phosphorylation occurs. Nevertheless, in the presence of ADP and phosphate. the high rate of electron transport is associated with a great deal of phosphorylation. We have called such oxidants Class 111 electron acceptors. Conventional hydrophilic oxidants such as methylviologen, ferredoxin-NADP‘ and ferricyanide we have called Class I acceptors. Since the reduction of Class III acceptors supports only half as much phosphorylation as the reduction of an equiva- lent amount of Class I acceptor, we suggested that lipophilic oxidants have access to 79 PHOTOSYSTEM ll l’lrlOSl’l lORYlATlON' 1“? and accept electrons l‘rom some electron carrier which lies between two sites of phosphorylation. Furthermore. we suggested that the second phosphorylation \lIC. the one not employed in the reduction ol' Class ill acceptors, is responsihle for limiting the rate of the liill reaction. Hence the high rate 01‘ ele ‘tron transport in the presence of Class lll acceptors. in the second paper4 we showed that KCN treatment ol‘ chloroplasts prevents the reduction ol‘ Class 1 acceptors but not the reduction of Class III acceptors. This virtually proves that Class lll acceptors do react directly with some :nter- mediate carrier in the electron transport chain, a carrier operating hel'ore the KC.\' block. Moreover we postulated that this intermediate carrier is close to Photo- system ll on the basis ofthe kinetics of DCM Ll inhibition ot‘the reduction ol‘ (‘lass lll acceptors. This in turn implies the existence of a r-Laispitorvlzttiott site closely asso- ciated with Photosystem ll. since the reduction of (fl.ts~— 1" ‘zt'c'cp'ufirs via the KCN- insensitive shortened pathway is still coupled with a l‘ e: ram ot‘ 0.3-0.4. In the present paper. we have shown that dihromothymmtuinone also inht its the reduction olClass l acceptors without severely inhibiting the reduction ol'Cla: lll acceptors. Regardless of the Class III acceptor used (oxidized [t-phenylenediamine. oxidized diaminodurene. oxidized diaminotoluene or 2.6-dimethyl-p-l‘enzoquinone) and therefore. regardless of the rate of electron transport and the P’e: ratio in the absence of the inhibitor, the dibromothymoquinone-resistant portion 01‘ electron transport is coupled to phosphorylation with a We; ratio of 0.35—0.45. A very similar P,’e2 ratio (0.3—0.4) is associated with the residual ferricyanide reduction in the presence of low concentrations ol‘ dibromothymoquinone. It is quite clear that all these reactions involve only Photosystem ll and that segment ol‘ the elecrron transport chain which ends in the dibromothymoquinone block. We must therefore conclude that there is a site of phosphorylation associated with Photosystem ll and located bel'ore the dibromothymoquinone inhibition site. ll' dibromothymo- quinone indeed blocks electron transport at the site of plastoquinone involvement, as the evidence suggests. there must be a site of phosphorylation both bel'ore and after plastoquinone. Thus the rate-limiting phosphorylation reaction presumed to occur between plastoquinone and cytochrome f“ may be equated to the slow step postulated in our lirst paper“. On the basis olcross-over point determinations, Bohme and CramerT concluded that only one phosphorylation site in the electron transport chain exerted a control over the rate of electron transport. However, our observations are in no way in- consistent with their conclusion. The transport 01‘ electrons to Class lll acceptors proceeds at high rates whether or not phosphorylation occurs and it is axiomatic that cross-over data cannot yield information on sites of phosphorylation unless the electron transport through the site is phosphorylation dependent. We have presented the bare bones of our conclusions in Fig. 5. No doubt alternative interpretations of the data could be devised but none has occurred to us. The precise location of Site 11. the site close to Photosystem ll which we have pro- posed in this paper, remains a matter (or conjecture. Neumann t'! u/.' 5 have provided a model of non-cyclic photophosphorylation in which two sites of phosphorylation are assumed to be involved in Photos}. tern l reactions. We (ind it dillicult to re- concile their model with our data unless ‘iite l in Fig. 5 is Further divided into two sites. lt is. however. possible that there is another site close to Photosystem l which 80 128 S. IZAVVA (‘I al. 02 90 i " Ol'e C. / fecy \ 98MB 4 . \/ I f*‘ecy izo-mosv Z—vo'otn—-cyii—~P;c—w=>5i tw . ocw paws sen \ADP ‘——v———’ ‘—v—’ SlTE ll SlTE I Fig. 5. Simplified scheme of the electron transport pathways. phosphorylation reactions and inhi- bition sites discussed in this paper. PD“. oxidized p-phenylencdiamine; Fecy, ferricyanide: DBMIB, 2.6-dibromo-3-methyl-6-isopropyl-p-benzoqtiinonc (dibromothymoquinone); PQ, plasto- quinone; PC. plastocyanin; DCM U, 3-(3.4-dichlorophenyl)-l,l-dimethylurea: MV, methyl- viologen; PS 1 and PS 11, Photosystenis l and 11. respectively. It should be noted that dibromo- thymoquinone and KCN both block reduction of the hydrophilic electron acceptors btit not the redtiCtion ol' the lipophilic acceptors. Moreover. the residual electron transport with either in- hibitor present supports phosphorylation with an etliciency tP/e: ratio) of 0.4. is responsible for some DCMU-insensitive cyclic photophosphorylation reactions, but this possibility is outside the scope of our present investigation. POSTSCRIPT After we had completed the manuscript ofthis paper we received a communica- tion from Dr Achim Trebst describing similar experiments conducted in his laboratory which have led him also to conclude that there is an energy conservation step associat- ed with Photosystem 11 reactions. ACKNOWLEDG EM ENT This work was supported by a grant, GB 22657. from the National Science Foundation, U.S.A. REFERENCES Winget, G. D., Izawa, S. and Good. N. E. (1965) Riot-Item. ”(tip/11V. Rm; Commun. 2!, 438—443 Izawa, S. and Good, N. E. (1968) Biochim. Biophys. Acta 162. 3804‘)! Saha, S., Ouitrakul, R., Izawa, S. and Good, i'. E. (1971) J. Bioi. C/tt’tn. 246, 3204—3209 Ouitrakul, R. and Izawa. S. (1973) Biochim. Biophys. Acta 305, lOS-llh‘ Avron, M. and Chance. 8. (1966) Brook/ture” Symp. Biol. ’9, l49-160 Kok, B., Joliot, P. and McGloin. M. P. H969) in Progress" in P/Itllt).\'}'NI/t(’.\('5' Research, pp. 1042—1056, lnternational Union 01‘ Biological Sciences. Tiibingen Bohme, H. and Cramer, W. A. (1972) Biochemistry 11. 1155—1160 Trebst, A., Harth. E. and Draber. W. (1970) Z. iV’tttttr/orse/i. 25b. llS7-1159 Bohme, H.. Reiner, S. and Trebst. A. (1971) Z. .N’utur/mst-li. 26b. 341—352 Bijhme, H. and Cramer, W. A. (1971) l-‘EBS Lett. 15, 349—351 11 Knall‘, D. B. (1972) FEBS Lett. 23. 142-144 12 Good, N. E. and Hill, R. (1955) Are/t. Biochem. Biophys. 57, 3554‘“) 13 Avron, M. (1960) [fine/tint. Biophys. Acta 40, 257-272 14 Lozier. R. H. and Butler, W. L. (1972) l-‘l-JIIS Lett. 26, 161—164 15 Neumann, J., Arntzen, C. J. and Dilley. R. A. (1971) Bioc/ieniistry 10, 866-873 outbuild— 'QWOONI APPENDIX II PHOTOSYSTEM II ELECTRON TRANSPORT AND PHOSPHORYLATION WITH DIBRONOTHYMOQUIN NE AS THE ELECTRON ACCEPTOR 82 Photosystem-II Electron Transport and Phosphorylation with Dibromothymoquinonc. as the Electron Acceptor J. Michael GOULD and Seikichi Izawa Department of Botany and Plant Pathology Michigan State University. Michigan (Received February Ill/April :26, 1973) Dibromothymoquinone has two effects on isolated chloroplasts. At very low concentrations it inhibits the reduction of conventional hydrOphilic electron acceptors, probably by acting as a. plastoquinone antagonist. At higher concentrations it acts as an electron acceptor, intercepting electrons either before or at the site of its inhibitory activity. Reduced t1ibromothymoquinone can be readily reoxidized by excess ferricyanide in the reaction mixture or by molecular oxygen. The transfer of electrons to this‘ substance from water is coupled to phosphorylation. The pH optima for this reduction and associated phosphorylation are both at 7.3, considerably lower than the pH optimum of 8.4 observed with the normal Ilill reaction. The ratio of molecules of ATP formed to pairs of electrons transported is relatively constant (0.3—0.4) between pH 6 and O. The rate of reduction is independent of the presence or absence of ADP and phosphate or uncouplers. The reduction of dibromothytnoquinone resembles the reduction of other lipid-soluble oxi- dants such as oxidized p-phenylenediarnines and 2,5-dimethquuinone in several respects. Both reactions are insensitive to the plastocyanin inhibitors KCN and polylysine, but are sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. They support phosphorylation with a similar efficiency. However, dibromothymoquinone reduction dichrs from the reduction of other lip0philic oxidants in that its sensitivity to dichlorophenyl-dimethylurea decreases with increasing light intensities. This implies that it may be reduced via a pool (plastoquinone?) which is a common electron acceptor for independent photosystem II units whereas oxidized p-phcnylencdiamines and 2,5- dimethquuinone are reduced directly by these independent units. The transport of electrons from photosystem II to photosystem I can be inhibited in several ways. Treatment of chlorOplast-s with KCN [1] or poly-L- lysine [2] seems to block the flow of electrons from cytochrome I to Pm, by inactivating plastocyanin [3,4]. In contrast, dibromothymoquinone probably acts as a plastoquinone antagonist [5] and therefore blocks electron transport at an entirely different site. These inhibitors abolish the transport of elec- trons from water to conventional hydrophilic electron acceptors such as methylviologen or ferredoxin- NADP+ but they do not prevent the reduction of lipophilic acceptors such as the oxidized forms of p-phenylenediamine and diaminodurene. Saha et al. [6] were the first to point out that these lipophilic “class III” oxidants could be reduced at Abbreviation. P/eg, ratio of the molecules of ATP formed to the pairs of electrons transported. Trivial names. Dibromothymoquinone, 2,5-dibromo-3- methyl-G-isopropyl-p-benzoquinone: dimcthylbenznqninone. 2,5 -dimethy1-p- benzoquinione; dichlorophenyl - dimethyl- urea, 3(3,4-dichlor0phenyl)-l,l-dimethylurca; I’mo. primary electron donor to photosystem I. exceptionally high rates by illuminated chloroplasts and that this rapid electron tranSport was coupled to phosphorylation. However, they also showed that the overall efficiency of phosphorylation, as measured by the ratio of molecules of ATP formed to the pairs of electrons transported (1’/e._. 5:; 0.5) is much lower than the efficiency with conventional hydrophilic "class I” acceptors (Pfegs 1.2). These findings led Saha el al. to postulate that class III acceptors might intercept electrons by reacting with an intermediate carrier situated between two sites of phosphorylation. The experiments with inhibitors alluded to above have strongly supported this concept. Ouitrakul and Izawa [1] have shown that the reduction of class III acceptors is largely insensitive to KCN treatment and Ort et al. [7] have shown that the same is true for polylysine treatment. Thus we may conclude that plastocyanin, which is required for the reduction of class I acceptors, is not required for the reduction of class III acceptors. Similarly we have shown that the plastoquinone antagonist dibromnthymoquinonc does not inhibit the reduction of class III acceptors 83 [8]. Consequently it seems likely that class III acceptors intercept electrons before the site of in- volvement of plastoquinone in the electron transport chain. Furthermore, since the reactions insensitive to dibromothymoquinone, KCN and polylysine con- tinue to support phosphorylation even in the pre- sence of these inhibitors, we concluded that there must be a site of phosphorylation associated with the photosystem II-drivcn transfer of electrons from water to plastoquinone [S]. In the course of our investigations we noted that, at concentrations somewhat higher than are required for inhibition of the reduction of class I acceptors, dibromothymoquinone itself acts as an electron acceptor [8,9]. Furthermore, the photoreduction of dibromothymoquinone was coupled to phosphoryla- tion with an efficiency characteristic of the reduction of class III acceptors. This paper deals with a more detailed investigation of the role of dibromothymo- quinone as an electron acceptor. The study has shown that this lipophilic oxidant has unique pro- perties which distinguish it from other class III acceptors. MATERIALS AND METHODS The procedures employed were similar to those described in previous papers [6,8]. Washed chloro- plasts were isolated from fresh market spinach (Spinacia oleracea L.) by ditferential centrifugation as detailed elsewhere [6]. Electron transport with ferri- cyanide as the electron acceptor was measured spectrophotometrically by continuously recording the decrease in absorbance of the reaction mixture at 420 nm. Electron transport with oxidised diamino- durene, dimethylbenzoquinone or dibromothymo- quinone as the electron acceptor was followed as the reduction of excess ferricyanide since the reduced acceptors were immediately rcoxid ized by ferricyanide present in the reaction mixture [6]. Oxygen uptake (Mehler reaction) was measured with a Clark-type membrane-covered oxygen electrode. No H202 trap was necessary since the chloroplast preparations were free of catalase activity. Saturating intensities of orange actinic light (> 600 nm) were supplied by a 500-watt slide projector and the appropriate colored filters. For some experiments the intensity of the actinic beam was varied with a series of calibrated neutral-density screen filters. Absolute light intensity was measured with a YSI radiometer shielded from infrared by a Corning heat filter ((3.8. 1—09). All reactions were carried out in thermostatted cuvettes at 19 °C. ATP formation was measured as the residual radioactivity remaining in the reaction mixture after extraction of the unreacted [32P]orthophosphate as phosphomolybdic acid [10]. Cyanidetreated chloroplasts were prepared as described by Ouitrakul and Izawa [I] by incubating chloroplasts at 0 °C for til) min in a 30 m.\I KCN solu- tion buffered at pH 7.8. Control chloroplasts for these experiments were incubated in a similar manner substituting KOII for KCN. Dibromothymoquinone was prepared as describct‘l earlier [8] and dissolved in ethanol—ethylene glycol (1:1,v/v). 3(1),4-Dichloro- phenyl)-1,1alirnethylurea was dissolved in ethanol and further diluted with 0.01 AI NaC'l. Stock solutions were prepared in such a way that the final concentra- tion of organic solvent in the reaction mixture never exceeded 1.50/0. Details of the treatment of chloro- plasts with poly-L-lysine (31,, 194000) are described elsewhere [7]. RESULTS Dibro 222 011i ymoqttinone- Catalyzed Reduction of Oxygen (.lIelzlcr reaction) In the absence of added electron acceptor, illuminated chloroplasts consume oxygen slowly (Fig.1). This phenomenon, which represents the reduction of small amounts of endogenous acceptors, their reoxidat ion by molecular oxygen, and the formation of llgO2 is known as the Mehlcr reaction [11]. There are two reasons for believing that the Mehler reaction catalyzed by endogenous acceptors involves photosystem I. The electron transport is coupled to plmsphorylation with an efficiency charac- teristic of the Hill reaction with elassI acceptors (l’/e._. a: 1.2) and treatments of chloroplasts with KCN or polylysine, which block the flow of electrons at plastocyanin [1 —4,] abolish the reaction. However, when dibromothymoquinone (15 pM) is added to the reaction mixture the rate of oxygen uptake is greatly increased and now neither KCN nor polylysine inhibits. The efficiency of phosphorylation also falls to the level characteristic of the reduction of class III acceptors (Table 1). It seems clear therefore that this dibromothymoquinone-cat.alj.-'zed Mchler reaction involves only photosystem II. Since dibromothymoquinonc is itself a powerful inhibitor of the transport of electrons to photosystem I, it totally suppresses the endogenous Mchlcr reac- , tion at low concentrations (Fig.2). As the concentra- tion of dibromothymoquinone is increased, the inhibitor begins to act as an electron acceptor, cat- alyzing the dibromotbymoquinone-I{C.\'-polylysine~ insensitive, photosystem II Mehlcr reaction. With the inhibition of the endogenous reaction the phos. phorylation efficiency (P,’c._.) promptly falls to a constant level oft).3 —O.4. It should be noted that this efficiency is quite independent ofthe rate of electron transport, depending rather on the electron transport pathway. Pnntrpl ACCED‘L‘Y EndegerCes M $55) 0 Nf‘ b 84 "CM - Heated (c l ‘3' M/WW‘W A Cata'ase seized Distomotrymcuuinm‘? if} \ \ 02 To: _ \l Uptake 'rLQQUiV. 1 +-—+i tain Catalase added Fig.1. The died a] K ON an endogenous and dibromothymo- quinone-catalyzed oyrgcn uptake (Mehler reaction) in illu- minated chloroplasts. The reaction mixture (2 ml) contained 0.1M sucrose, 50 m3! tricine bufl'cr pll 8.1, 2 mM MgClz, Other Characteristics 0/ the Electron Transport and Phosphorylation Associated with Dibromothymo- quinone Reduction The unusual dual action of dibromothymoquinone, inhibiting the transport of electrons to photosystem I while at the same time serving as an electron acceptor, makes its reduction a convenient reaction for the study of photosystem II. However, the reoxidation by oxygen becomes seriously rate-limiting below pH 8.0 or when the concentration of the inhibitor- aeceptor is lowered (Fig.2. This difficulty can be overcome by the addition of ferricyanide since it has already been shown that ferricyanide rapidly re- oxidizes reduced dibromothymoquinone [8]. Indeed we have shown that this chemical reoxidation by ferricyanide is not rate-limiting under any of the conditions employed in this study; the rate of dibromothymoquinone reduction, measured as the consequent reduction of excess ferricyanide, is con- stant with time until virtually all of the ferricyanide has been reduced. Since the dibromothymoquinone- catalyzed reduction of oxygen and of ferricyanide both support a phosphorylation reaction having the same efficiency and the same sensitivity to photo- system I inhibitors (Table 1), it seems probable that the two reactions are biologically equivalent. For these reasons, and because less dibromothymoqui- none is required to catalyze ferricyanide reduction, we used the dibromothymoquinone-ferrieyanide sys- tem in the experiments described below. The pH optimum for photosystem II electron transport and phosphorylation (Fig.3) seems to be considerably lower than the optimum observed for the Hill reaction with class I acceptors (7.3 vs 8.4). This lower pH optimum observed in the presence of 0.5 mM ADP. 5 mM P3, and chloroplasts containing 40 ug chlorophyll. When added. a small amount of catalase was injected into the sample chamber with a. mieroliter syringe. Numbers in parentheses are. acquiv. x h‘1 :< mg chlorOphyll‘l i); C) l P’Cz () in _._,.. \ O ._._..._... C '1‘ ‘3 L E C. r— ‘I O 3,. )3 ”I“ ‘- l" ~' c- U ' . I A (Pn_ IDIbrCTPOZHymoquincre} (LL-l) .- s w a o t O 8. u‘) C: S E '50-— _ '3 3 l C it / / 0V l‘.) :3 3'3 [Dit'cmcthys‘oquinorei turd) Fig.2. E/[ect of dibromothymoquinonc on electron transport (Ind phosphorylation in the absence of nth/cc! electron acceptor. Reaction conditions were as in Fig. 1. Electron transport (0) was measured as oxygen uptake as described in Methods. (0) ATP formation. (0) Pfcg. Units are as in Table 1. Note that low concentrations of dibromotliymoquinone abolish the endogenous Alehler reaction (I’,e2 :1 1.1). Higher concen- trations catalyze a photosystem II Mehler reaction which supports phosphorylation with a l’,"e2 of about 0.4 dibromothymoquinone. cannot be an effect of pH on the phosphorylation mechanism since the same optimum is seen in the presence or absence of ADP 85 Table 1. Effect of photosystem-I inhibitors on electron. transport and pr}u/op/msplioryluIion. wit/1 eurfous clrtctrrm acceptors The reaction mixture (2 ml) contained 0.1 M sucrose, 51) 11131 1ri11i:111-.\'.-11)ll pH 14.1. :3 m.“ Muti‘ly 0.5 mM ADP, 5111311 Kali:- 331’04, chloroplasts containing 411 341.: ehlm'oph3'll and the indicated acceptor syszem. in 1311- rlihrouiothymorpnnone to ferricyanide (DBMIB —> I'eCy) system the rem-tion was liuil'ered at pH 7.5 with 5.1111.“ .1\'-2-l1_1'1!r1).\_1'111l13'lpi1.11:1/.i111.1-.\"-'.’-ethanesulltmnte. The acceptor systems used were: Fet'y. 11.4 n1.\'1 ferricyanide: l’lJM. 1.1.5111.“ p~pl11~11yi1111111li:1111i1111 plus 1.5111.“ ferricyanide; DBMIB -> 03. 15 1.131 dibromothyinmi 11ino11e; l)l‘ 3“ H -+ FeCy, HD1131 diiimiuot113'1111'11111i11111111 plus 11,-} 2n.\l ferricyanide. I\C.\'- treated chloroplasts were 1111 p;1r1d as 111-.e1r1111d in Methods. l’oi3'-1..l_1'.~1i111--11112111111 chloroplasts were prepared as detailed by Ort ct al. [7]. Rates of 11icctro11 tr::1n.11port (if. '1. l and pl10~1phur3'l:11i11:1 (.-\'l‘l’) are given as 11.11q11i1'. or gunol ATP >’ h" :/ mg: chlorophyll“. Note that the reduction 01‘ the class III acceptor l’l ‘M has :1 cyanide and 110i3'l3'si11c-sensiti111. component. Since dibromothymoquinone blocks the transt'e 1r of electrons to ]}l1()if1$\nictll 1.31s rcd11c1inn l.--11l;s this 1-.11111pone11t Note also that the residual photosystem II electron tram-ppm supports phosnhor1'1.1tion mm .111 11l'il 1 i11111v (1’ 1.1 _.) oi 1hout 11. ‘.l—-—H t. 'Jhe lower efficiency after pol3'l3 sine treatmcntot the e. 1l11ropl: 131s is probahh due .1) the 1111M 111111111uncouplingelk-1:1 or this substance ~1.11] 11111:: r1111 :11'1'1'111111‘ “NM-1111 _. ._-_,,-__,_____ -.—-.—...- ____, E. __._.__..__ -_.... £3} Condition }1'c(‘_v 111104.. 1:11:11) 1111111111-. 0,) DBMIB (—. FcCy) F. T. A'l‘l‘ (l’,’c.) E. '1'. ATP (l'le,) l1}. '1'. ATP (P/e.) E. T. ATP (Pica) I control 401 253 (1.213) 11.170 310 (0.5.“ 171” 20 (0. ' 37) 262 50 (0.3.9) KC.\' 35 5 (~) 1311:. 108 (0.31;) 141; 17 (11; 2:11 2:1”: 4:1 (11.31%) H control 5130 347 (1.24) 1301) 42‘.) (0.61) 110 21') (t). '3131 275 43 (0.31) polylysine 20 0 (—) 980 218 (0.213) 116 1‘.) (11.5. ) 2b?) 36 (0.25) 0.1. _ I I 1 1 _1 Table 2. The lack 0/ film! 0/ phosphate, ADIP or uncouplers o 9 a I on Iii/iron:wiry/110711{none redurlion O/O’A A ““9\ The basic reaction mixture (pH 7.5) was 11.11 in Table I. The ‘3 0 2_" _ acceptor system used was 101.131 dibromothymoquinone ' plus 11.4 mM, i'1-1-rie3'1111i1le. Additions were made in small volumes of distilled water (11.1)?» ml) to the final concentra- O 1 1 1 , tions indicated below: ADP, 11.5 111.“; 1’1. 5111M; methyl- 50 ' I l ' amine l13'1ir01-i1loride, 11) 111.“; gramicidin I), 4;.12/111l l 11— 1—1 ' l'lxpcriiuent. Electron 0’ A No. Additions transport rate a. 111~1111iv.;vh“'x111g chlorophyll" Q - "‘ I None 2.11 ‘09 ‘1 .1 1111 1.1.1:: ADP + 1’: - graim- cidin I) 221 Electron 1ransport (11/2) or ATP II ADP «I- P: _.1() ADP «j— 1’; + metl1yl~ amine 253 II None 204 so —( 3 .11111 17 P1 _ 21.15 /‘./_‘\! DD\ :/'/ !\- D\ -\ 1 1 '1‘ O 6 7 8 9 independent of phosphorylation. It should also be pH noted that the phosphorylation efficiency with Fig.3. E/lect of pH on dibromothymoquinone reduction nrul dibromothymoq11inone as the electron acceptor is associated phosphorylation. The basic rcz11tion mixture used alynost independent of pII over a, “ride range. This is is described in Tablel.1011\1dibromothymoquinoneplus in striking contrast to the cll‘ect of l‘” on the 0.4 mM ferricyanide was the acceptor system. The bullers 1 (50 mM) employed were (. 0’0) 2_ (V. inorpiiolino)etl.- ”3' phosphorylation cl'ticiencv when class I acceptors sulfonate, (A, A, A) N- 2 -h3dr0\3ethylpiperazine \’ MC reduced (Optimum PH: 31"“ 0) [12] ethanesulfonate, and (I, D, [2) tri1ine. Lnits for electron transport (II) and phosphorylation (111) are giequiv or 11111101 . . ATth‘lxmg chlorophy'll 1. Note that the efficiency. We. EH?“ 0] Dec/iloroplmnyl-dtmethylated (I), of the phosphor3 lat1on is almost, independent of DH on Dibromothymoqumone Photorcductzon from (5 to 9 . . . . Although dibrmuothymoqumone reduet1on 18 not affected by electron transport inhibitors acting close and phosphate: indeed the rate of electron transport to photosystem 1 (Table 1), it is sensitive to inhibitors during the reduction of dibromothymoquinone such as 231-72 hcpt3l- 4- hychoxyquinoline-N- oxid1 (Table 2) or other class III acceptors [6] is quite (Gould and 1511111131111, unpublished ohseimtions) and '7; 1C0 l— , --------- E r" O/ 3 3 1' ' .«2 ca °~ .C U c I I7) \ '3: 50 L: a L'- 400 bc) .‘3 :/ _. ' C t ’7 \ C I o z: o "‘ no ; 9’ *3 0‘5 1 1 g 3 C 0.1 O .2 )C .3 3' RCMUlt .. 200‘; 5 .1 %' Ro E \ c \. 9. '6 .\\ .93 u l o \6': J O 01.2 0.4 [DCMUI(uM) Fig.4. Inhibition by dichlorophenyl-dimethylurco of electron transport with various acceptors. The basic reaction mixture is described in Table 1. When ferricyanide (0.4 mM) was used as the electron acceptor (O) the reaction das butl'ered with- 50 mM tricine-NaOH pH 8.1. When dibromothymoquinone. (10 nM plus 0.4 mM ferricyanide) was the acceptor system (0), the reaction was bull'ered with 50m)! V-. " hydroxy- ethylpiperazine-i ’-2-ethanesull‘onate pll 7.5. (-----) lie- duction of oxidised diaminodurene from a separate experi- ment [1]. Note that at the high light intensities employed the photosystem II reduction of dibromothymoquinone and the reduction of ferricyanide have the same sensitivity to dichlorophenyl-dimethylurea (DCMU) while the reduction of oxidised diaminodurene is much more sensitive dichlorophenyl-dimethyl urea which block the elec- tron flow close to photosystem II. Fig.4 shows the effect of the latter substance on the rate of dibromo- thymoquinone reduction. This exhibits about the same sensitivity to dichlorophenyl-dimcthylurea inhibition as does the reduction of the class I acceptor ferricyanide. Dibromothymoquinone differs from class III acceptors in this respect, since the reduction of the oxidation products of p-phenylenediamine and diaminodurene is extremely sensitive to concen- trations of dichlorophenyLdimethylurca which have very little effect on ferricyanide reduction [1]. The effect of light intensity on dichlorophenyl- dimethylurea inhibition of electron transport with various electron acceptors is shown in Fig.5. As the light intensity becomes rate-limiting, dibromo- thymoquinone reduction and ferricyanide reduction become more sensitive to this inhibition, while the high dichlorophenyl-dimethylurea sensitivity of the reduction of oxidised diaminodurene remains un- changed. Again dibromothymoquinone behaves more like a class I acceptor than a class III acceptor under these conditions. 86 A l '3 . 0: l l c \ *3 ------------- a -------- ---s--- a E ’ -: .40 \°\o\\ 0"— u— ‘_ ~— -‘ ~0— rOO — -" \ 400 ‘5 a — J \ >- 10 1:3 v/I 200 _. C l 1:) 15 V/I Fig.5. The c//cct of light intensity on dichlorophcnyl-Ilimethyl- urea inhibition of electron transport supported by various acceptors. {eat-tion conditions were as described in Fig.4. (a, A, A) 0.3 in.“ diaminodurene dihydrochloride plus 1.5 mM ferricyanide as electron acceptor, (O. O, O) ferricyanide re- duction and (D, l, [2) dibroinothymoquinone reduction. Solid symbols, 0.075 31..“ dichlorophcnyl-dimcthylurca added. (B) e represents the rate of electron transport in uequiv. X h"1 X mg chlorophyll'1 and 1 represents the light intensity in kerchm‘ZXs“. The intercept on the c-axis represents the rate of electron transport extrapolated to inlinitcly high light intensity (V) while the intercept on the t'jI-axis gives the quantum efficiency extrapolated to zero light intensity. Note that the primary ell'ect of dichlorophenyl-dimethyl- urea is to lower the quantum efficiency, presumably by blocking independent photosystem [I chains (see Discussion). Dibromothymoquinone also has a pronounced secondary eil‘ect on the quantum efficiency. Note also from (A) however, that the inhibition by dichlorophcnyl-dimcth_vlurea decreases with increasing light intensity when ferricyanide or dibromo- thymoquinone are reduced but not. when oxidised diamino- durene is reduced These differences in sensitive to dichlorophenyl- dimethylurca and the echcts of light intensity on the inhibition could be explained in two ways: either the high rates involved in reduction of oxidised diamino- durene make the dichlorophenyl-dimcthylurea site more critically rate-determining or oxidised diamino- durene intercepts electrons close to the site of di- chlorophenyl-dimethylurca inhibition (see Discus- sion). The experiment illustrated in Fig.6 all but eliminates the first possibility. The photoreduction C0 ‘4 ”30 I l I \r l y 3 so '0 G o _o_ l ‘.: 1 - 9_'i 3 a a 2 — .2 C O l l I l o 53 it.) ~=e \’ .acc -? .1 Light intensity (Lev; . cm - s ) T l 8 101A'— -( o 8 500 '— -—t l. \ o \‘. o I. \ 30 ‘5‘ ‘\\ O 1 \ ‘1‘ \ O 5 10 15 v/[ Fig.6. El/ect of light intensity on dichlorophenyhdimethylurea inhibition 0/ dimethylbenzoquinone reduction. The basic reac- tion mixture (pH 8.1) is described in Table 1. Diuiethylbenzo- quinone (0.5 mM) plus 0.4 in.\l ferricyanide was the acceptor system. (0, 0) No additions; (O, I) 0.075 p.31 (llClllOl’O- phenyLdimethylurea added; ([1, (2, I) 0.5 u.“ dibromothymo. quinone added, sufficient to block electron flow to photo- system I but not high enough to accept electrons at a signif- icant rate [3]. Note that the dichlorophenyl-dimcthylnrea inhibition of dimethylbenzoquinone reduction is independent of light intensity both in the presence and absence of di- bromothymoquinone. Note also from (B) that the. latter has a secondary effect on the quantum efficiency of this reduc- tion of 2,5-dimethylbenzoquinone has two components. One component is sensitive to KCN, polylysine and low concentrations of dibromothymoquinone [8]. This component clearly involves both photosystems and is in every way analogous to the reduction of class I acceptors. The other component is insensitive to these three inhibitors and supports phosphoryla- tion with the efficiency characteristic of the reduction of class III acceptors. Clearly dimethylbenzoquinone intercepts some electrons in the same manner as oxidised p-phenylenediainine and diaminodurene, but it does so slowly. Thus the residual transport of electrons to dimethylbenzoquinone in the presence of low concentrations of dibromothymoquinone is approximately equal to the rate observed when di- bromothymoguinone itself is used as an electron ac- ceptor. Nevertheless dimethylbenzoquinone reduction remains more sensitive to dichlorophenyl-dimethyl- urca than is (ii'ormnothymoouinone reduction and this Sensitivity does not. vary with light intensity. It should be added here. that the quantum efficiency of dibromothymoquinone reduction (i.e. dibromo- 1hymoqninone-mediatcd fcrricyanidc reduction) is only boo/0 of the normal ferricyanide IIill reaction (Fig.5B). Apparently dibroniotilyinoquinone has a rather strongr secondary clT‘ct on the quantum efficiency of photosystem ll (see also FigJiB). '. ‘his is also reflected in the fact that at very low light intensities dibrmnothyinoquinone reduction becomes even more sensitive to dichlorophcnyl.dimethylurca than does the reduction of oxidised diaminodurene (Fig.5A). DISCUSSION Dibromothymoquinone as an Electron Acceptor The fact that KCN and polylysine treatments of chloroplasts have no effect on the rate of electron transport (Table 1) virtually proves that photo- system I is not involved in the reduction of dibromo- thymoquinone since these treatments block the trans- f'ir of'clectrons to l.’.,oo{.‘l, 4]. Moreover dibromothymo- quinone is itself a potent inhibitor, blocking electron transport on the photosystem II side of cytochrome I [14]. It follows that dibromothymoquinone must accept electrons either before or at its own site of inhibition. In some respects dibromothymoquinone reduction resembles the reduction of other lipophilic quinones and quinonediimides (class III acceptors) which also accept electrons from a carrier close to pliotosystem II (1,6,8). However, the reduction of dibromotliymoquinonc is unlike the reduction of class III acceptors in other respects. For instance, the concentration of dibromothymoquinone which is optimal for electron transport is 50 times lower than the optimal concentration of oxidised diaminodurene or p-phenylencdiaminc. This observation could be interpreted as implying some degree of specificity but it could also be interpreted in terms of a partition coefficient which favors accumulation of dibromo- thymoquinone in the membranes. A much more important difference which distin- guishes dibromothymoquinone from class III accep- tors is the. naturi- - " the inhibition of its photoreduc- tion by dichlorop‘.:nyl-dimcthylurca. The effect of this compound on dibromothymoquinone reduction is similar to the efl‘ect on the reduction of ferricyanide (class I) and is quite different from the effect on the reduction of oxidized p-phenylcncdlamine and di. aminodurene (class III). The much greater sensitivity of chloroplasts to low concentrations of dichloro- phenyl-dimethylurca with class III acceptors (Fig.4, inset) can readily be explained in terms of the model presented in Fig.7. This model also explains the fact that dichlorophenyl-dimethylurea inhibition of elec- PHCTOSYSTEM II UNtTS 88 A r W ClossllI oc~iu Acceptors DBMIB (high) H 01:21:. ‘ >l——_l/ 2 . . 8 )_._,----- Hzoi::__—3—_(> o L -+—#------ H20c::t:> ; (fl----.I H20 :1) (pc 7) : HZOt_—_;_r—_’:;’> ~ SlTE II Fig.7. A simplified model of electron transport in chloroplasts. The open arrows represent functional structures each of which contains pigments and a reaction center of photo- system II, a single oxygen-producing site, and a site sensitive to dichlorophenyl«limerhylurea (DCMU). The straight DBMS t'ow) -;-——/\NV\»—>cyf. f—j—oPS I —-> ’ Class L Acceotors KCN POLYLYSlNE ~ SITEI arrows represent electron transfer reactions and curved arrows represent energy conservation reactions (phosphoryla- tion sites). The zig-zagged line at site I indicates the primary rate-limiting step of electron transport to class I acceptors. For further explanation, see the text. DBMIB = dibromo- thymoquinone tron transport is independent of light intensity when class III acceptors are reduced but becomes intensity- dependent when class I acceptors are reduced (Fig. 5). There are good reasons for believing that the photochemistry and much of the associated thermo- chemistry of photosystem II takes place in indepen- dent structural units [15]. In other words, it seems that quanta are converted one by one into charge sepa- rations within independent structures. Thus the elec- trons and holes made available by quantum conver- sions in one structural unit are notd ircctly available for chemical reactions in other units. We have represent- ed these water-oxidizing, electron-producing photo- system II units in Fig.7 by the open arrows. While the exact nature of dichlorophenyl-dimethylurea’s inhibition is not clear, it seems likely that it acts by somehow inactivating photosystem II units, one molecule of inhibitor totally suppressing the activity of one unit. A partial inhibition by dichlorophenyl- dimethylurea probably means that a certain propor. tion of the photosystem II units have been inactiv- ated. ‘ . However, there is no reason to suppose that photosystem I must be. confined to the same unit structures as photosystem II. Consequently it is not unreasonable to suppose that the electrons generated by photosystem II may be peeled at some step before the reduction of P700. Indeed one might expect reduc- ed plastoquinone to serve as a common electron pool interconnecting electron transport chains on the basis of its chemical nature and its abundance. In fact, Siggel et al. [16] and Malkin and Michacli [13] have reached this conclusion from their flash experi. ments and fluorescence induction studies using chloro. plasts poisoned with dichlorophenyl-dimethylurea. “’6 are now in a position to understand the effect of light intensity on dichlorophenyl-dimethylurea inhibition. When light is limiting, the activity of photosystem II is presumably also limiting and the inhibition will be strictly proportional to the number of units inactivated by dichlorophenyl-dimcthylurea. The same will be true, regardless of light intensity, if the electrons are never pooled. However, if the elec- trons arc pooled and the pooled electrons are utilized by a subsequent ratealetermining slow step, the situ- ation is quite different. Now a smaller number of func- tioning photosystem II units can keep the slow reac- tions draining the electron pool saturated. Thus, as the light intensity increases, fewer and fewer photo- system II units are required and the efficacy of a given concentration of dichlorophenyl-dimethylurea decreases. These considerations suggest to us that class III acceptors are reduced before the electrons from photo- system II are pooled [1]. Presumably these membrane. permeating Strong oxidants react directly with the photosystem II units. Clearly ferricyanide and other class I acceptors must be reduced after the electrons are pooled since their reduction is less sensitive to dichlorophenyl-dimethylurca at high light intensities. Although dibromothymoquinone is highly lipid- solublc and is a moderately strong oxidant, it does not seem to react directly m'th photosystem II units but rather with the source of pooled electrons. We are tempted to postulate that dibromothymoquinone, by virtue of its structure, reacts in some specific way at the site of plastoquinone involvement in electron transport, accepting electrons from reduced plasto- quinone and at the same time blocking further transport of electrons to cytochrome /. It is not immediately clear how the model present- ed in Fig. 7 can be reconciled with the experiments of Izawa and Good [19], which suggest that the number of dichlorophenyl-dimethylurea inhibition sites is smaller than the number of 0.2-producing units (1 0: producing unit per 500 chlorophylls [15]). “’0 are now inclined to (pies-tion the interpretations oll‘ered by Izawa and Good for their observations. Photosystem. II I V: as phorylation. with Dibromothymoqu[none as 15h (sh-on Acceplor The data presented in this paper strongly support our contention that there is a phosphorylation reac- tion associated with the transfer of electrons from water to plastoquinone [8). Moreover it is very probable that the energy conservation occurs before the electrons produced by individual photosystem II units are pooled. If this is so, each of the units must be equipped with an appropriate erici‘g__ty-con.scrvin;r mechanism (site II in Fig.7). Perhaps the photo- system II units are so oriented that the photoaetiva- tion or subsequent electron transport occurs across the thylakoid membrane and the resultingr membrane potential or ion gradient drives phosphorylation, as Witt [17] has suggested. On the other hand the as-yet unassigned cytochrome bm may be associated with photosystem II units and may be involved in some chemical mechanism of energy conservation. In any event, photosystem II phosphorylation is very‘clitferent from the better-known phosphoryla- tion associated with the reduction of class I acceptors, both in efficiency and in pH dependence. The lower efficiency with which photosystem II electron trans- port is coupled to phosphorylation may simply result from the fact that only one of two (or more) in a sequence of phosphorylation sites is being.' used (0). On the other hand the complete lack of dependence of electron transport on phosphorylation may reflect. a fundamentally inefficient coupling mechanism which in turn may reflect a basic difference between the oxidation-reduction reactions performed at site II and site I. Indeed, the insensitivity to pH of the phosphorylation efficiency at site II (Fig.3) is in striking' contrast to the pH dependencve of the Pb: attritmtable to site I (12). The authors nish to thank Dr N. 17.. Good for many valuable. criticisms during the preparation of this manuscript. 'l‘he studies reported here were supported by a grant (t i ii 226.37) from the Xatinnal Science Foundation, U.S.A. ll lll"l".l‘.l7..\'ClCS l. Ouitrakul. 11. a; lzawa, S. (1973) [Hoe/tint. Biophys. Add, .305. IDS—l IN. -. lrand. J.. Baszynski, T.. Crane, l“. & Krogmann. D. (l‘JTL‘) J. Biol. Claw)". “347, 2514—3310. I}. Izawa. 5.. Kraayl‘nhul'. ll., l—iuugc. E. K. & DcVault, D., Biochim. Biol-flute. A ('11:. in press. 4. Brawl, J., San Pietro, A. & Mayne. B. (1972) Arc/i. Biochem. lilo/Jigs. IJL’. 43.3—42.4. 0. Biiiime. ll.. lleiiner.S. &. 'l'rcbst,.-\. (1971) Z. Natur- IIOI'SI‘II. 'l'r’il If. 26!). 341—352. (3. Saha, S., Ouitrakul, IL. lzawa, 9. &. Good. N. E. (1971) J. Biol. Chem. L’I/J, Iiflll-‘t—il'lllll. 7. Ort, l). ll., lyzawa. 8., (Mod. X. lC. (1973) H's/1s Mr. 31, 119-122. S. Izawa, S., Haniltl. J. 33., Urt, D. l“... Feikcr, P. 8!. Good, X. 1'3. (19723) line/rim. Biophys. Acta, {fl/5, 119—128. 9. Louier. l1. ll. .'\' Butler, W. l.. (1072) I’ll/3S Lelt. :30, Hi] — 1134. l“. Avron, )l. (1960) Biochim. h'opliys. Acta. 40, 257—272. 11. )lehler. A. H. (1951) .‘ll‘f'll. Bier/mm. Biophys. 33. 65—77. 2. Winget. (l. 1)., Izawa, S. & (100d. N. E. (1965) Biochem. lite/21:31.3. li'es. Commun. :3], 4238—443. 13. Malkin. S. & Michaeli. L}. (1972) in Proc 2nd Int. Congr. on [’lmtosynthesis Res. Ntrrsa. (Forti, 0., Avron. M. &. Melanrlri,.-\. eds.) Vol. 1, p. 149-107, Dr W. Junk N. V. Publishers, The Hague. l4. Bohme, H. & Cramer, W. A. (1971) FEBS Lett. 15, IH‘. —':;--)l. 1.7. Kok. R., Forbush. l3. & MeGloin, M. (1070) Photochem. l’lunlom'ol. II, 457 ~-4-7.';. ll}. Siggel. U., Renger, (1., Stiehl. ll. H. & Rumberg. B. (1972) Bier/rim. Biophys. Acta, 350, 3228—335. 17. \Vitt. ll. T. (1971) (,2. Rev. Hinpliys. ~I. 365—477, 18. Diilcy. '1. .-\. (10053) Biochemistry 7, 338—346. 19. Izawa. S. a (500d, N. 1'}. (100.3) Biochim. Biophys. Acta, 103, Qll—BS. St Krugmann, D. W. J. M. Gould and S. Izawa, Department of Botany and Plant Pathology, 166 Plant Biology Building, Michigan State University, East Lansin" \lichigan 4mm}, U.S.A. 59‘ APPENDIX III STUDIES ON THE ENERGY COUPLING SITES OF PHOTOPHOSPHORYLATION I. SEPARATION OF SITE I AND SITE II BY PARTIAL REACTIONS OF THE CHLOROPLAST ELECTRON TRANSPORT CHAIN Reprinted from Biochimica et Biophysica Acta, 314 (1973) 211—223 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 46 589 STUDIES ON THE ENERGY COUPLING SITES OF PHOTOPHOSPHORY- LATION I. SEPARATION OF SITE I AND SITE II BY PARTIAL REACTIONS OF THE CHLOROPLAST ELECTRON TRANSPORT CHAIN J. MICHAEL GOULD and s. IZAWA Department of Botany and Plan! Pathology, Michigan State University, East Lansing, Mic/I. 48 823 (U.S.A.) (Received April 9th. l973)_ SUMMARY 1. The transport of electrons from H20 to lipophilic oxidants such as oxidized p-phenylenediamines and 2,5-dimethquuinone, when observed in the presence of the plastoquinone antagonist dibromothymoquinone, has a pH optimum of approxi- mately 7.5 and is independent of the presence or absence of ADP and phosphate. Nevertheless the electrontransportsupports phosphorylation with an efficiency (P/ez) of0.3-0.4andthisefliciencyis practically pHindependent.A reversible proton uptake is associated with the electron transport. The energy coupling site responsible for the phosphorylation, which must be before plastoquinone, we have designated Site ll, while the well-known rate-determining coupling site after plastoquinone and before cytochrome/is referred to as Site I. 2. The transport of electrons from reduced 2.6-dichlorophenolindophenol (DCIP) to methylviologen, when observed in the presence of the Photosystem II inhibitor 3-(3,4—dichlorophenyl)-1,1-dimethylurea, is remarkably similar in most respects to the overall Hill reaction (e.g. H20»methylviologen). The rate of electron flow is markedly stimulated by ADP and phosphate. Electron transport and phos- phorylation have the same pH optimum of about 8.5. The P/ez ratio is also strongly pH dependent, showing a similar pH optimum of 8.0-8.5. However, the absolute value of the P/ez ratio observed for the partial reaction reduced DCIP—>mcthylvio- logen is lower than the P/e2 ratio observed for the overall reaction H20—>methylvio- logen at all pH values. The maximum P/e2 value observed for the reduced DCIP» methylviologen raction is 0.5—0.6 at pH 8.0-8.5 while the maximum value for the HID->methylviologen reaction under the same conditions is about 1.1. 3. When the P/ez ratios for the two partial reactions (HZO->dimethquuinone and reduced DCIP—avmethylviologen) are added together at all pH values from 6 to 9, the resulting curve is very close to the P/ez-pH profile experimentally obtained for Abbreviations: P/ez. the ratio of the molecule: of ATP formed per pairs of electrons transported; 4'-deoxyphlorizin, 4,6’-dihydroxy-2'-glucosidodihydrochalcone; DCIP, 2,6—dichloro- phenolindophenol; DCMU. 3-(3,4-dichlorophenyl)-l,l-dimethylurea; Tricine, N-tris(hydroxy- -ethyl)methylglycine; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesult'onic acid. 92 212 J. M. GOULD. S. IZAWA the overall Hill reaction HZO->methylviologen. It seems probable, therefore. that the transport of electrons from reduced DCIP to methylviologen utilizes only the rate- determining coupling Site I while the overall transport of electrons from H30 to methylviologen utilizes both Site I and Site ll. . INTRODUCTION The existence of two sites of energy coupling associated with noncyclic electron transport in isolated chloroplasts has been postulated for some time based on various lines of indirect evidence"7. A new approach to this problem has recently been made possiblewiththe use ofthelipophilic “Class III" electron acceptors (such as oxidized p-phenylenediamines)“. In previous papers we have shown that thetransportofelectrons from waterto these Class III acceptors is insensitiveto the plastocyanin inhibitors KCN9 and poly-L-lysine‘o, and also to the plastoquinone antagonist dibromothymo- quinone”. These inhibitors strongly inhibit the reduction of conventional (Class I“) acceptors such as ferricyanide or methylviologen, which require participation of both Photosystem II and Photosystem I. Furthermore, Class lll acceptors are reduced at a point before the electrons from the independent Photosystem ll units are pooledg'”. From these observations we have concluded that the reduction of Class III acceptors takes place predominately before plastoquinone”. Moreover. since the redUCtion of Class III acceptors is firmly coupled to phosphorylation even in the presence of these inhibitors, we have concluded further that there is a coupling site before plastoquinone (Site II) in addition to the coupling site believed to be located after plastoquinone and before cytochromefl3 (Site I). The phosphorylation reaction associated with Site II is characterized by (i) a pH optimum between 7 and 8, (ii) a low coupling efficiency (P/cz =0.3—0.4) which is practically pH independent, and (iii) the lack of effect of ADP and phosphate or uncouplers on the rate of electron transport. The characteristics of conventional noncyclic photophosphorylation, which utilizes both Site II and Site I, are quite different. The pH optimum for both electron transport and ATP formation is 8.5 or above. The phosphorylation efficiency is also a strong function of pH, showing a similar optimum (where' the P/e2 is l.O—-l.l in average chloroplast preparations). Furthermore, the rate of electron transport responds sharply to phosphorylating or uncoupling conditions. One may therefore reasonably deduce that these prominent features of conventional noncyclic photophosphorylation originate almost entirely fromthecoupling reaction at Site I. The existence of a rather inconspicuous coupling reaction at Site II must be largely masked, except for its contribution to the overali efficiency of phosphorylation. In order to verify these deductions, however, it is essential to find a partial reaction of the electron transport chain which includes only coupling Site I. Such a reaction should very much resemble the complete noncyclic reactions except for its efficiency of phosphorylation (P/ez), which should be approximately 0.6—0.7, instead of slightly above 1.0. , Larkum and Bonner“ and Izawa", on the basis of spectral evidence. and Neumann er al.“5 on the basis of uncoupler studies, have postulated that reduced 2.6- dichlorophenolindophenol (DCIP) in the presence of 3-(3,4—dichlorophenyl)-!,l- dimethylurea (DCM U) donates electrons to the electron transport chain on the Pho- 93 [J ,1 SITES OF ENERGY COUPLING IN CHLOROPLASTS tosystem Il side of the coupling site preceeding cytochrome f. The studies reported in this paper have provided strong evidence that the electron flow from reduced DCIP to methylviologen indeed constitutes a partial reaction which includes coupling Site I but not coupling Site II. In addition, we report here and in a subsequent paper:7 on further characterization ofthe coupling reaction at Site II including the demonstra- tion of a "proton pump" driven by a partial reaction which involves only Site ll. MATERIALS AND METHODS Chloroplastswereisolated in the cold (4 °C) by a technique similar to that used in previous studies‘” "“2. Leaves of fresh market spinach (Spinacia oleracea L.) were washed in cold distilled water and ground brielly (3-7 s) in a Waring blendor con- taining a medium consisting of 0.3 M NaCl. 30 mM N—tris(hydroxyethyl)methyl- glycine (Tricine)-NaOH (pH 7.8). 3 mM MgCl2 and 0.5 mM EDTA. After filtering the homogenate through multiple layers ofchecsecloth, the chloroplasts were sedimen- ted at 2500 wig for 2 min. The pellet was resuspended in a medium containing 0.2 M sucrose, 5 mM .V-Z-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)- NaOH (pH 7.5), 2 mM MgCl2 and 0.050;, bovine serum albumin. After a brief cen- trifugation to remove whole cells and debris (45 s at 2000-(g) the chloroplasts were sedimented at 2000 :« g for 4 min, resuspended in a fresh volume of the same medium, and again sedimented. The final pellet was taken up in a small volume ofthe suspen- sion media. The reduction of 2.5-dimethyl-p-benzoquinone, oxidized p-phenylenedi- amines and high concentrations of dibromothymoquinone was measured spec- trophotometrically as described earlier” as the decrease in absorbance of the reaction mixture at 420 nm due to the reduction of excess ferricyanide. Methyl. viologen reduction (either water or artificial reductants as electron donor) was mea- sured as oxygen uptake resulting from the reoxidation of reduced methylviogen'8 A Clark-type membrane-covered oxygen electrode was used for assay. No catalase. inhibitor was needed since our chloroplast preparations were free of catalase ativity. Reactions were run in a final volume ol'2.0 ml in thermostatted cuvettes at l9 C. Actinic light (>600 nm) was supplied by a SOO-W slide projector and the appropriate lilters. ATP formation was determined for a l-ml aliquot of the reaction mixture by extracting unreacted “P-Iabeled orthophosphate as phosphomolybdic acid into butanol-toluene (lzl, v/v) as detailed by Saba and Good”. Radioactivity in the final aqueous phase was measured by the Cerenkov technique of Gould er ol. 2". KCN-treated chloroplasts were prepared by incubating chloroplasts at O "C in a 30 mM KCN solution bulIered at pH 7.8 as described by Ouitrakul and Izawa". Stock solutions of 2.5-dimethyl-p-benzoquinone and dibromothymoquinone were made in ethanol—ethylene glycol (l :1. v/v). DCIP was dissolved in ethanol and diluted with glass distilled water. DCMU was dissolved in ethanol and further diluted with0.0l M NaCl.Atalltimestheconcentration oforganic solvent in the final reaction mixture was I92, or less. RESULTS The effect of pH on the rate of electron transport and phosphorylation using three different electron donor-acceptor systems is shown in Fig. I. When electrons 214 J. M. GOULD, S. IZAWA 800 1 I If t I fl r t t t I DCIPHz—vmv H20—-OMQ soo- / «- «~ i ET. 0 o E.T. (‘8') (t p.) r ET. I’Pi) ATP \ /O-\° I 7 / ELECTRON TRANSPORT or ATP Ir" : 0 a o, 0/0 ET ”’0 I3/ / \o {'O/ D\< Ofl-D-‘O A \ 2°°' "/| " /a//o/O 'f‘I/ ATP ‘ I ’0 ' a p’s/o \ :31: A 'fIOP.\...-... "-'!‘.....‘~. :9—049/ Ute.) ,.-- .x" ‘ -Q---b" 1 1 L An-A- ‘1 1 1 f l 1 1J 6 7 8 9 5 7 8 9 6 T 8 9 p H Fig. I. Effect of pH on the rates of electron transport and phosphorylation associated with various electron donor-acceptor systems. The reaction mixture (2 ml) contained 0.1 M sucrose, 2 mM MgCl-g, 50 mM buffer, 0.75 mM ADP, 5 mM Na-zHiV-‘P04 (when added), chloroplasts containing 40 pg chlorophyll, and the indicated electron donor or acceptor system. These systems were: methylviologen, 50 pl“: 2,5-dimethyl-p-benzoquinone, 0.5 mM plus 0.4 mM ferricyanide; reduced- DCIP (DCIPHg), 0.4 mM plus 2.5 mM ascorbate, I/lM DCMU and 50;:M methylviologen. The buffers employed were 2-(.V-rnorpholino)cthanesulfomc aetd—NaOH (triangles), HEPES- NaOH (squares) and Tricinc~NaOH (circles). Open symbols are for electron transport (E.T.) and solid symbols are for phosphorylation (ATP). When 2,5«dimethyl-p-benzoquinone was the electron acceptor 0.5;:M dibromothymoquinone was added to the reaction mixture to block the Class 1 component of 2,5-dimethyl-p-bcnzoquinonc reduction. Rates are in [ICQUIV or ”moles ATP/h per mg chlorophyll. Note the similarity between H:O->methy1viologen and reduced DCIP»methylv)oltigen but not HzO-r2,5-dimethyl-p-benzoquinone.Abbreviations: MV,methyl- viologen; DCIPHz, reduced DCIP; DMQ, 2,5-dimethyl-p-benzoquinone. from water reduce the Class I acceptor methylviologen through the two sites ofenergy coupling, both electron flow and phosphorylation show a pH optimum around pH 8.5. The rate of electron transport in the absence of phosphate (basal rate) is much slower but shows a similar pH optimum. This is in good agreement with previous reports for ferricyanide reduction and the associated phosphorylation'. When reduced DCIP serves as electron donor and methylviologen as acceptor(in t he presence of DCM U) the effect of pH is very similar to that observed for the HzO—wmethylviologen reaction. Again the optimal pH for electron transport and phosphorylation is about 8.5 and a marked stimulation of electron transport by the concomitant phosphorylation is observed. Clearly the two reaction systems H20--methylviologen and reduced DCIP amethylviologen are governed by the same rate-limiting phosphorylation reaction. However, when the Class III acceptor 2,5-dimethyl-p-benzoquinone is reduced w’o Photosystem II by electrons from water, a different effect of pH is evident. 2.5-Dime- thyI-p-benzoquinone reduction and the associated phosphorylation (which involves only coupling Site II) exhibit a considerably more acidic pH optimum than is observed for the Hzoemethylviologen system or the reduced DCIP—>methylviologen system. The effect of pH on the phosphorylation efficiency (P/ez) of each of the three types of electron donor—acceptor systems mentioned above produced some striking results (Fig. 2). The P/e2 ratio for the HzO—e-mcthylviologen system is strongly pH 95 SITES OF ENERGY COUPLING IN CHLOROPLASTS 215 l l l T (e) HZO-c-HV (SITE 101!) ° r—o I.O— '\/ \o\— / , "I" “ ‘ ~ ~. ,’ tomb) \\ , \ I l I :1 (a) OCIPHz—OMV (Sl‘TEII E . .\ 0-5‘ ./ o\ -i / x a O‘LLO-o_° o I A /’ lb) nzo—uouo (SITE in A 6 7 8 9 pH Fig. 2. Effect of pH on the phosphorylation efficiency (Plea) of three different electron donor— acceptor systems. P/ez values for each system were computed from the data presented in Fig. 1. Note that if the We: values for H20»2.5-dimethyl-p-benzoquinone (Photosystem II only) are added to the values for reduced DC1P~>methylviologen (Photosystem I only), the sum (dashed curve) is close to the P/(: values obtained for HzO—«methylviologen (Photosystem 11 plus Photo- system 1). Note also that the P/e: ratio associated with 2.5-dimethyl-p-benzoquinone reduction is practically constant over a wide pH range, whereas the We: ratios for H 20 +methylviologen and reduced DCIP~>methylviologen are strongly pH dependent. Abbreviations: see Fig. 1. dependent, showing a pH optimum of about 8 to 8.5. In contrast, the P/e2 ratio for the H2092.5-dimethyl-p-benzoquinone system, which utilizes only coupling Site II is essentially pH independent from pH 6.5 to 9. Thus the pH-dependent portion of the P/ez ratio for the HZO—>methylviologen system must be located after the site of 2,5-dimethyl-p-benzoquinone reduction. The effect of pH on the P,"e2 ratio associated with the reduced DCIP-+methylviologen reaction once again resembles very closely the effect observed for the Hzoamethylviologen system. This similarity again sug- gests that the coupling site associated with the reduced DCIP—>methylviologen system may be the same rate-limiting coupling site (Site 1) associated with the H20—>methyl- viologen reaction. However, the P/e2 values observed for the reduced DCIP,—>methyl- viologen system are markedly lower than those for the H20»methylviologen system over the entire range of pH values tested, as though a pH-independcnt component is absent in the reduced DCIP system. Indeed, when the P/e2 values for the two partial reactions (HID—>2,5-dimethyl-p-benzoquinone and reduced DCIP-+methylviologen) are added together, the resulting curve (Fig. 2, dashed line) is in fact very close to the experimentally obtained curve for the overall reaction H20»methylviologen. This implies very strongly that the partial reaction reduced DCIP—smethylviologen is indeed utilizing only the coupling site that normally limits the rate of electron transport to Class I acceptors (i.e. Site I). The curve derived by adding the P/e2 values for the two partial reactions is 96 216 3. M. GOULD, S. IZAWA slightly lower than the observed curve for H,O~>methyi\iologen, reficCting the fact that the PM; ratios observed for the reduced DCIP~.~methylviologen reaction (maxi- mum 0.55) are slightly lower than the values one would expect (0.6—0.7) from the difference in P/e2 between the complete system HzO+methylvioiogen and the partial system H20->2.5odimethyI-p-benzoquinone. This discrepancy could be explained in two ways. If reduced DCIP had a secondary, uncoupling elf: t the P,’e2 values for the reduced DCIP—>methylviologen reaction would be lowered. This seems unlikely. however,sinceit has already been shown that the reaction exhibits a comma: P/‘ez over a wide range ofreduced DCIPconcentrations“.To furthereliminatethis possibilitythe effect of reduced DCIP on the post-illumination phosphorylation ("XE") was examin- ed. lf reduced DCIP had an uncoupling action, then its presence in the dark (phos- phorylation) stage of the experiment should decrease the yield of X5. The high sensi- tivity of this method in detecting an uncoupling effect has been previously demon- strated by Hind and Jagendorf“. Table I shows that neither reduced DCIP nor 2.5- dimethyl-p-benzoquinone decreasesthe XE yield to any significant extent, thus prac- tically eliminating the possibility ofthese compounds having a significant uncoupling efTeCt. These results are also important in that they ensure that the low efliciencies of phosphorylation observed for systems involving 2,5-dimethyf-n-benzoquinone (P/ez=0.3—0.4) or reduced DCIP (P/ez=0.5—0.6) are not due to an uncoupling effect of these compounds. TABLE I EFFECTS OF REDUCED DCIP PLUS ASCORBATE AND 2,5-DIMETHYL-p-BENZO- QUINONE ON POST-ILLUMINATION ATP FORMATION (xii) Reduced DCIP (0.I3 mM plus 0.8 mM ascorbate), 2,5-dimethyl-p-benzoquinone (0.17 mM) or methylamine (3.3 mM) were present only in the dark phosphorylation stage of the experiment. Chloroplasts containing lOO/ig chlorophyll were illuminated for 20 s in a continuously stirred reaction mixture (2 ml) containing 0.1 M sucrose, 50 mM NaCl, 2 mM MgClg, 10 mM 2-(N-mor- pholinokthanesulfonic acid-NaOH buffer (pH 6.0) and SiiM pyocyanine. Immediately after shutting off the light 1 ml of a strongly buffered ADP-phosphate mixture (0.1 M Tricine-NaOH buffer (pH 8.2), 2 mM ADP, l0 mM Na2H32PO4) containing the additions was quickly injected into the suspension to initiate ATP formation. After 20 s the dark phosphorylation was terminated by addition of 0.5 ml 1 M HCIOi. All reactions were run in a thermostated water bath at l9 ‘ C. Note that both reduced DCIP (plus ascorbate) and 2,5-dimcthyI-p-benzoquinone did not inhibit the yield of X2, whereas the known uncoupler methylamine did. Addition Exp! ATP formed Ellt'c‘! (dark stage) (nmoles/ lO’J my chlorophyll ) None a 7.4 -— b 7.7 Reduced DCIP a 9.2 Slight stimulation b 8.4 2,5-Dimethyl-p-benzoquinone a 6.9 None b 7.5 Methylamine a 2.6 Inhibition b 2.4 97 SITES OF ENERGY COUPLING IN CHLOROPLASTS 217 TABLE II EFFECT OF KCN TREATMENT ON ELECTRON TRANSPORT AND PHOSPHORY- LATION ASSOCIATED WITH THE PHOTOSYSTEM I-DEPENDENT REACTION RE- DUCED DCIP—eMETHYLVIOLOGEN Reaction conditions are as described in Fig. 1. KCN treatment of chloroplasts is described in Methods. The rates of electron transport (E.T.) and phosphorylation (ATP) are given in pequiv or umoles ATP/h per mg chlorophyll. Electron transport from H20 to methylviologen in the KCN-treated chlorOpIasts used here was completely inhibited. Reaction Control chloroplasts K C N-treated chloroplasts H p E. T. A TP E. T. A TP 6.0 l 27 4 9O 2 7.0 240 49 l 23 5 8.0 397 102 1 54 4 9.0 420 93 148 I Alternatively, some of the electrons from reduced DCIP may be donated to Photosystem I via a secondary, nonphosphorylating pathway. In fact, there is already strong evidence for this possibility. Ouitrakul and IzawaQ have shown that the phos- phorylation associated with the reaction reduced DCIP»methylviologen is abolished by KCN treatment but the electron transport itselfis only partially inhibited”. Recent- ly, Izawa or al.“ have shown by EPR studies that reduced DCIP can donate electrons directly to P700, by-passing a KCN block at plastocyanin. They suggested that this portion of the donor reaction is not coupled to phosphorylation. Table II shows the effect of KCN treatment on the electron transport from reduced DCIP to methyl- viologen and the associated phosphorylation. Undoubtedly the reduced DCIP—rmeo thylviologen reaction contains a minor component which is KCN resistant and not coupled to phosphorylation, (It should be noted here that KCN treatment itself has no appreciable uncoupling or inhibitory effect on phosphorylation?) It therefore seems reasonable to conclude that the “true" PM: values for this partial reaction are slightly higher than those shown in Fig. 2 (curve it). Thus the true sum of the Pie, values obtained for the partial reactions reduced DCIP->methylviologen (involving Site I only) plus HzO- ’2.5-dimethyl-p-benzoquinone (involving Site II only) must indeed be very close to the values obtained for the overall reaction HzO—imethylviologen (Site II plus Site I). The conclusion that the complete noncyclic electron transport HZO—>methyl- viologen and the partial electron transport reduced DCIP—>methylviologen are gov- erned by the same energy coupling reaction (Site I), is further strengthened by the experiments of Fig. 3 in which the effects of the energy transfer inhibitor 4’-deoxyphlo- rizin“ on electron transport and phosphorylation were examined. In both systems ATP formation and that portion of the electron transport which is dependent upon the presence ofADP and phosphate are inhibited in a very similar manner. However, the phlorizin derivative has no effect on electron transport from H20 to oxidized diaminodurene (a Class III acceptor3)either in the presence or absence of phosphate, although phosphorylation is inhibited. These observations are directly in line with the concept that the coupling site near Photosystem II (Site II) has no control over 98 218 J. M. COULD, S. IZAWA [ T T I Y fi j T j I ‘I'x/E‘T m" DCIPH -—-—Mv ‘ H o--0A0 ' (SITE ii I ('5le iii ' Hzo—oiviv I I l Ag _ I (Sire-ion) l -,* = l 2 a. 750t- ' x + .,‘ p. T ET IOHA) I O v I ¢—o_° 0"- i p. l a I . / l c. 500r- I T [7 up.) 'i V) ‘2‘ I rt? (oP') (. [[1 (.P') 1' t: o F I at (-P-) I\/ E ”"3 t I z ' I \ O a o / I 0% .I s was x Nexe i . .———-—-2: 1 I 8 a . I 3, ATP I " I “‘ ATP/\a_ I\ _,/’"p ;\‘./ Q. ' 1 ‘\A ‘ I \A _ 1 o i s - 1 . C‘PD 4-DEOXYPHLORIZIN (M x to“) Fig. 3. Effect of the energy transfer inhibitor 4'-deoxyphlorizin on electron transport and phos- phorylation associated with different electron donor—acceptor systems. Reaction conditions are essentially as described in Fig. l. The buffer used was 50 mM Tricine—NaOH (pH 8.0). Methyl- amine (MA) was I0 mM when added. The chlorophyll concentration was 20/ig/ml. When oxidized diaminodurene (DADM) was. the electron acceptor the chlorophyll was long/ml and 0.5/4M dibromothymoquinone was added to blocx the Class 1 component of oxidized diaminodurene reduction. Rates of electron transport (E.T.) and phosphorylation (ATP) are in ,ieuuiv or iimoles ATPfh per mg chlorophyll. Now that for H20 —>methylviologen and reduced DCIPHmethy-l- viologen both ATP formation and that portion of the electron transport dependent upon phos- phorylation are inhibited by 4’-deoxyphlorizin. However, when the (Iiass ll! acceptor oxidized diaminodurene is being reduced, eleCtron transport is not affected by the absence of phosphate or the presence of 4’-deox_vphlorizin, although phosphorylation is inhibited by the latter. Also note that ATP formation associated with all three electron donor-acceptor systems exhibits about the same sensitivity to 4'-deoxyph|orizin. Abbreviations: see Fig. I. electron transport while the site between plastoquinone and cytochrome f (Site 1) does. Relevant to these fiindings is the question of why the reduction of oxidized p-phenylenediamines (typical Class III acceptors) did not seem to be stimulated by ADP and phosphate at all“, despite the fact that some portions of these acceptors are reduced via the complete electron transport pathway (as are Class ! acceptors). utilizing both coupling sites (Site II and Site I)“. We have reinvestigated this problem and found that the concomitant phosphorylation does stimulate electron flow quite consistently (Table III). The stimulation may seem quite small, but this is simply because the very fast reduction of these compounds by Photosystem II. The absolute stimulation is in fact approximately equivalent to the stimulation observed when electrons flow from water to methylviologen. However, when this “Class 1 component" ofthe reduction of Class III acceptors is eliminated by the addition oflow concentra- tions of dibromothymoquinone (or by KCN treatment"). the electron transport. now mediated only by Photosystem II and utilizing only coupling Site ll. becomes com- pletely independent of phosphorylating conditions. The genuine Photosystem ll eleCtron transport from HzO to dibromothymoquinone (high concentration) is also not influenced by phosphorylation (Table III‘, or by-uncounlers‘z. 99 SITES OF ENERGY COUPLING IN CHLOROPLASTS 219 TABLE III EFFECT OF PHOSPHORYLATING CONDITIONS ON ELECTRON TRANSPORT AS A FUNCTION OF THE ELECTRON ACCEPTOR The 2.0-ml reaction mixture contained 0.1 M sucrose, 2 mM MgCl:. 1 mM ADP, 5 mM Na2H33P0¢ (if added‘, 50 mM Tricine-NaOH (pH 8.1), chloroplasts, and the indicated acceptor. These acceptors were: methylviologen, 50,1434: oxidized p-phenylenediamine, 0.5 mM plus 1.5 mM ferricyanide; dibromothymoquinone, 4.5iiM plus 0.4 mM ferricyanide. The concentration of chlorophyll was 20/igyml when methylviologen or dibromothymoquinone acted as the electron acceptor and lS/ig;ml when oxidized p-p‘nenylenediamine was the electron acceptor. Rates are given in pequw or iimoles ATP/h per mg chlorophyll. Nete that the reduction of oxidized p-phenylenediamine contains a phosphate-sensuive Class 1 component. If this component is abolished with a low concentration of dibromothymoquinone (which blocks electron transport to cytochrome [3") the effect of phosphate on oxidized p-phenylenediamine reducuon is elimi- nated, even though a substantial rate of phosphorylation remains. Similarly, the Photosystem II- driven reduction of high concentrations of dibromothymoquinone, which accepts electrons from plastoquinone”, is firmly coupled to ATP formation even though the eleCtron transport shows no effect by phosphate. u“- Electron transport system Acceptor C (mp/ing si'tc Electron transport rate class ' involved ' " ‘* -P. + Pt /.1 ET. ATP Plea HzO—emethylviologen I Site ll 4- Site I I90 458 (268) 276 1.20 Hzo—eox. pphenylenediaminc III (+1) Site ll (+Site I) 1370 1560 (I90) 405 0.52 H20->OX. p-phenylenediamine (+5' 10—7 M dibromothymoouinone) Ill Site II 860 860 (O) l85 0.43 0.34 H20—rdibromothymoquinone Ill Site II 193 200 (2) 34 - -V..._._ --- ~.—-—-u-—.— ' See ref. 8 and Introduction. Proposed sites of phosphorylation (see Fig. 5 and refs 9, ll, l2). Fig. 4 shows a light induced pH rise (“proton uptake“) associated with coup- ling Site II when 20 [AI dibromothymoquinone was the electron acceptor. It can be seenthat at pH 8.1, where reduced dibromothymoquinone is rapidly reoxidized by molecular oxygen”, repeated periods of illumination induced the familiar reversible pH rise in the reaction medium. If DCMU is added (abolishing eleCtron transport) the pH rise is also abolished. Similary, at pH 7.4 a briefiilumination induces the pH rise. However, at this pH reduced dibromothymoquinone is net reoxidized by molecular oxygen. Thus, repeated illuminations do not induce a pH rise once all ofthe dibromo- thymoquinone is reduced and electron transport cannot proceed. This is comfirmed by the fact that addition of oxidized dibromothymoquinone restores the pH rise. The Photosystem Il-dependent pH rise is also abolished by conventional uncouplers such as methylamine and gramicidin. DISCUSSION In previous papers°'“'” we have amply documented evidence that there is a site of energy coupling (Site 11) near Photosysrem II or, more specifically, before plastoquinone, in addition to the well-recognized coupling site (Site I) which lies be- tween plastoquinone and cytochrome f and governs the rate of noncyclic electron lOO 220 ‘ J. M. GOULD, S. IZAWA A 0) initial pH 8.! 083,-, l i i l W $ 1 1 4 1 4. ‘. l LIGHT on -l T LEGHT OFF-( 30 nequiv b) initial pH 7.4 ADD 1- DBMIB i i l i I 4 l t i |——Ol i5 see Fig. 4. Light-induced pH rise in the medium (“proton uptake") associated with the reduction of dibromothymoquinone by Photosystem II. The continuously stirred sample was illuminated in a thermostatted cuvette (l9 "C) with strong white light. The change of pH of the medium was recorded Using a Sargent miniature combination electrode and a Corning expanded scale pH electrometer. The scale was calibrated by adding in the light a known amount of HCl. The 2.0-ml sample contained 0.1 M sucrose, 2 mM MgCl2, 50 mM NaCl, 20/tM dibromothymoqiiinone. 0.5 mM Tricine«NaOH bulTer at the indicated pH and chloroplasts containing 140 [lg chlorophyll. When DCMU was added (Expt a) the Iinal concentration was 2.1 ,uM. In Expt b 4?) nmolcs dibromothymoquinone (DBMIB) were added to restore the initial pH rise. flow‘3'z‘. The main body of evidence comprises the fact a unique type of phosphory- lation is observed when the lipophilic Class III oxidants (e.g. 2.5-dimethyl-p-benzo- quinone, oxidized p-phenylenediamincs, etc.) are reduced i-i'a Photosystem II alone. To observe these genuine Photosystem II reactions. the simultaneous reduction ofthe acceptors through Photosystem lneeds to beeliminated (a) by blocking plastoquinone with dibromothymoquinoneu"26 or (b) by inactivating plastocyanin with KCN“ '0'” or (c) by using dibromothymoquinone itself as a Class III acceptor (at high concentra- tions)”. The phosphorylation elliciency (P/ez) of these Photosystem Il reactions is always between 0.3 and 0.4, regardless of the acceptdr used"'”"2 and therefore regardless ofthe electron transport rate, which ranges from about 50 ,uequiv/h per mg chlorophyll when dibromothymoquinone is used as the acceptor to about 1500 when oxidized p-phenylenediamine is the electron acceptor. (This makes it extremely un- likely that the phosphorylation associated with the reduction of Class III acceptors is due to a Photosystem II-catalyzed cyclic electron flow.) The efficiency of phosphory- lation supported by Site II is practically independent of pH over a wide range (ref. l2; see also Fig. 2, this paper). The electron transport is not stimulated by ADP and phosphate (ref. 12; see also Table III, this paper). When the electron transport chain is not blocked at plastoquinone or at plastocyanin, that is, when Class III acceptors are being reduced in part by Photosystem II alone and in part via both Photosystem II and Photosystem I, then the characteristics of the associated phosphorylation reac- tions become intermediate between those outlined above and those observed for lOl SITES OF ENERGY COUPLING IN CHLOROPLASTS 221 standard noncyclic photophosphorylation reactions sum as with ferricyanide or methylviologen (Class I acceptors). There seems no doubt that we have succeeded, by the combined use of Class III acceptors and the inhibitors which block electron trans- port at or after plastoquinone, in disclosing and functionally “isolating" a coupling site (Site ll) located before plastoquinone. In this paper we have presented strong evidence which shows that the Photo- system I-dependent transport of electrons from reduced DCIP to methylviologen involves only Site I (Figs l—3). The noninvolvement of Site ll in this reaction is also consistent with the fact that the reaction is totally insensitive to dibromothymo- quinone”. The lack of dibromothymoquinone inhibition indicates that plasto- quinone, and therefore the coupling site before piastoquinone (Site If). does not participate in the reduced DCIP—»methylviologen partial reaction. Photosystem l-dependent reactions with reduced DCIP as electron donor have often been regarded as complex, involving a cryptic cyclic phosphorylation which could make phosphorylation appear completely unrelated to the observed rates of electron flow“. However, at least under the conditions employed in this study, the relation of phosphorylation to observed electron flow seems quite rigid, judging by the ef‘fectof ADP and phosphate. the uncoupler methylamine. and the energy transfer inhibitor 4'-deoxyphlorizin. Trebst and Pistorius” have presented brief data which led them to the same conclusion. Neumann et al.”’ have postulated, based on their uncoupler studies,that the reduced DCIP-smethylviologen reaction and the HzO—p methylviologen reaction share the same rate-limiting phosphorylation'site (our Site I). Shavit and Shoshan28 have pointed out that high concentrations of ATP (where ATP acts as an energy transfer inhibitor) affect the reduced DCIP~2~NADP* system and the HZO—>NADP* system in a very similar manner. However, a detailed comparison of the phosphorylation reac'tions associated with the reduced DCIP system and the complete noncyclic system, such as we have presented here, has not been previously reported. The failure of earlier investigators to detect significant phosphorylation asso- ciated with the photooxidation of reduced DCIP by Photosystem I may have been due largely to the nature ofthe chloroplast material used. Swollen, broken, or other- wise “leaky” chloroplast preparations give a high rate of electron transport (reduced DCIP—>methylviologen) with very little. if any, phosphorylation. This electron trans- port is mostly KCN’ insensitive (unpublished data of J. M. Gould) and therefore probably represents increased access of reduced DCIP directly to P700 (ref. 23). Larkum and Bonner“ have found that the reduced DCIP-induced cytochrome f response is also greatly diminished in broken chloroplasts. The main conclusions we have drawn from this study are summarized in the scheme presentedin Fig. 5. When Class I acceptors (e.g. methylviologen) are being reduced by electrons from water or reduced DCIP, the rate ofelectron flow is limited by the energy coupling reaction between plastoquinone and cytochrome f (Site I). The electron ffuxthrough Site I responds strongly to the addition of ADP and phos- phate, uncouplers, or energy transfer inhibitors. The efficiency of phosphorylation (P/ez) at this site is also pH dependent. having a maximum at pH 8.0-8.5 (observed maximum P/ez, 0.5—0.6; predicted. 06—057). As mentioned above, Site If exerts no apparent control over electron flow. The efficiency of phosphorylation at Site II (P/e2=0.3—0.4) is essentially pH insensitive. One possible explanation for this pH 102 222 J. M. GOULD, S. IZAWA CLASS III acceptors DCIPHZ DBMIB lhith HZO-OPS II —-Po E-chytf—r Pic *Pm'PS 1" chcigisoé ~' patina ~ xcn (low) SITE II SITE I Fig. 5. A scheme for electron transport pathways in isolated chloroplasts showing the two sites of energy coupling (~). PS II, Photosystem II; DBMIB, dibromothymoquinone; PQ, plasto- quinone; cyt f, cytochrome /; PC, plastocyanin; P700. primary donor to I’hOtosystem I (PS I). The zig-zagged line at Site I represean the primary rate-limiting step of electron transport to Class I acceptors. Class I acceptors include methylviologen, ferricyanide, flavins and ferredoxin- NADP". Class III acceptors include oxidized p-phenylenediamine, oxidized diaminodurene, 2,5-dimethyl-p-benzoquinone, etc. Since Class III acceptors can also act to some extent as Class I acceptors, genuine Photosystem II nations are observed only when plastoquinone or plasto- cyanin is blocked (by dibromothymoquinone or KCN, respectively). Thus, in the presence of these inhibitors, the partial reaction H20—>Class III acceptor includes coupling Site II but not Site I. Similarly, the partial reaction reduced DCIP—+Class I acceptor includes coupling Site I but not Site II, whereas the overall reaction Hzo—rClass I acceptor includes both Site II and Site I. Note also that the reduced DCIP—»Class I acceptor contains both a KCN-sensitive and KCN-insensitive component. insensitivity could bethat coupling Site II is buried in a hydrophobic region ofthe mem- brane and therefore does not “see” the medium pH. This is consistent with the idea put forth by Bdhme and Trebst" and Yamashita and Butler7 that there is a coupling site on the water oxidizing side of Photosystem II, perhaps associated with the water- splitting reaction itself. It has been suggested that the protons lost during the oxida- tion of water are released to the inside of the thylakoid”. H,0-+2e‘ +«l02 +2H5,,,d,, The pH rise observed in the external medium when dibromothymoquinone (high con- centration) is the electron acceptor (Fig. 4) would therefore represent the loss of pro- tons from the medium for the reduction of the lipophilic dibromothymoquinone 2e' + dibromothymoquinone +_2H{f,,,,,,d,,-+reduced dibromothymoquinone in the thylakoid membrane. It is therefore probable that a transmembrane proton gradient is associated with the pathway HanPhotosystem II-odibromothymo- quinone as well as with reduced DCIP—>Photosystem I—>methylviologen“. Finally, it should be mentioned that the relationship between Site I (see Fig.5) and the two coupling sites postulated by Neumann et al.16 to be associated with Photosystem I is still unclear. Preliminary experiments have indicated that ATP for- mation coupledtothe Photosystem I-dependent electron flow from diaminodurene to methylviologen may not utilizethe coupling site which limitsthe H20 (or reduced DCIP) amethylviologen reaction (unpublished data of J. M. Gould). Experiments are in progress to determineiffurther subdivision ofSitel into two sites may be necessary. 103 SITES OF ENERGY COUPLING IN CHLOROPLASTS 223 ACKNOWLEDGEMENTS The authors wishto thank Mr Don Ort and Dr N. E. Good for valuable discus- sions and Dr G. D. Winget for the gift ofthe 4’-deoxyphlorizin. Supported by a grant (GB 22657) from the National Science Foundation, L'.S.A. REFERENCES Vic-wu— _oooeqos I} I4 15 l6 l7 I9 20 22 23 24 25 26 27 28 29 30 31 32 Winget, G. D., Izawa, S. and Good, N. E. (I965) Biochem. Biophys. Res. Commun. 21, 438—443 Izawa, S. and Good, N. E. (1968) Biochim. Biophys. Acta I62, SRO-391 Schwartz, M. (I968) Nature 2|9, 9I5-9I9 West, K. R. and Wiskich, J. T. (1973) Biochim. Biophys. Acta 292, I97—205 Hall, D. O., Reeves, S. G. and Baltscheffsky, H. (l97l) Biochem. Biophys. Res. Commun. 43, 359-366 Bohme. H. and Trebst, A. (I969) Biochim. Biophys. Acta I80, l37—I4S Yamashita, T. and Butler, W. L. (I968) Plant Physiol. 43. 1978-4986 Saha, S., Ouitrakul, R., Izawa. S. and Good, N. F.. (l97l) J. Biol. Chem. 246, 3204-3209 Ouitrakul, R. and Izawa, S. (1973) Biochim. Biophys. Acta 305, IOS—I 13 Ort, D. R., Izawa, S., Good, N. E. and Krogmann. D. W, (1973) FEBS Lett. 31, 119—122 Izawa, S., Gould, J. M., Ort, D. R., Felker, P. and Good, N. E. (1973) Biochim. Biophys. Acta 305, Il9—I28 Gould, J. M. and Izawa, 5. (I973) Eur. J. Biochem., in the press BOhme, H. and Cramer, W. A. (I972) Biochemistry II, I ISS-I I60 Larkum, A. \M D. and Bonner, W. D. (1972) Biochim. Biophys. Acta 26", l49-IS9 Izawa, S. (I968) in Comparative Biochemistry and Biopi‘iystcs of Photosynthesis (K. Shibata, A. Takamiya, A. T. Jagendorf and R. C. Fuller, eds). pp. I40-I47, University Park Press, State College, Pennsylvania Neumann J., Arntzen, C. J. and Dilley, R. A. (I97l) Bloc/tentislry l0, 866-873 Bradeen, D. A., Gould, J. M., Ort, D. R. and Winget, G. D., Plant Physiol., submitted for pubhcadon Izawa, S., Connolly, T. N., Winget. G. D. and Good, N. E. (I966) Brookhaven Symp. Biol. I9, 169—I84 Saha, S. and Good, N. E. (I970) J. Biol. Chem. 245, 50I7—502I Gould, J. M., Cather, R. and Winget, G. D. (I972) Anal. Biochem. 50, $40-$48 StrOtmann, H. and von Gosseln, C. (1972) Z. Naturforsch. 27b, 445-455 Hind, G. and Jagendorf, A. T. (I965) J. Biol. Chem. 240, 3202-3209 Izawa, S., Kraayenhof, R., Ruuge, E. K. and DeVault, D. (I973) Biochim. Biophys. Acta, in the press Avron, M. and Chance, 8. (I966) Brookhaven Symp. Biol. 19, I49-I60 B'o'hme, H., Reimer, S. and Trebst, A. (1971) Z. Naturforsch. 26b, 34I~352 TrebSt, A. and Reimer, S. (l973) Biochim. Biophys. Acta, submitted for publication Trebst, A. and Pisotrius, E. (I967) Biochim. Biophys. Acta ISI, 580-533 Shavit, N. and Shoshan, V. (I971) FEBS Lett. I4, 265-267 Junge, W., Rumberg. B. and Schroder, H. (I970) Eur. J. Bloc/rem. 575. I4 BGhme, H. and Cramer, W. A. “W” FEBS Lett. I5, 349-351 Winget, G. D., Izawa, S. and Good, N. E. (1969) Biochemistry 8. 2967—2074 Avron, M. and Neumann, J. (1968) Annu. Rev. Plant Physiol. i9, l37—I66 APPENDIX IV STUDIES ON THE ENERGY COUPLING SITES OF PHOTOPHOSPHORYLATION III. THE DIFFERENT EFFECTS OF METHYLAMINE AND ADP PLUS PHOSPHATE ON ELECTRON TRANSPORT THROUGH COUPLING SITES I AND II IN ISOLATED CHLOROPLASTS TOS Reprinted lmm Bloc/tiniica et Biophysical Acta, 325 (I973) l57—l66 g,“ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 46624 STUDIES ON THE ENERGY COUPLING SITES OF PHOTOPHOSPHORY- LATION III. THE DIFFERENT EFFECTS OF METHYLAMINE AND ADP PLUS PHOSPHATE ON ELECTRON TRANSPORT THROUGH COUPLING SITES I AND II IN ISOLATED CHLOROPLASTS ‘ J. MICHAEL GOULD and DONALD R. ORT Department of Botany and Plant Pathology, .N’Il'C/llg'llll State University, East Lnnsini', .llic/t. 48824 (U.S./f.) (Received June 4th, I973) SUMMARY 1. The reduction of lipophilic (Class III) oxidants such as oxidized p-phcnyl- enediamine consists oftwo components. One component requires both Photosystem II and Photosystem I and includes both sites of energy coupling associated with non- cyclic electron transport. The second component requires only Photosystem II and includes only the site ofenergy coupling located before plastoquinoiie (Site ll). When oxidized p-phenylencdiamine is being reduced by both pathways. the overall rate of electron transport is stimulated by the addition of ADP plus phosphate or the un- coupler mcthylamine. However. if the Photosystem 1 component of oxidized p- phenylenediamine reduction is eliminated by a low concentration of the plasto- quinone-antagonist dibromothymoquinone. the stimulation of electron transport by ADP plus phosphate or methylamine is also abolished. although the remaining Photo- system Il-depcndent electron transport remains firmly coupled to phosphorylation (via coupling Site 11). These results indicate that coupling Site If, unlike the well- known rate-limiting coupling site between plastoquinone and cytochrome/(Site I), does not exert any control over the rate of associated electron transport. 2. When substituted p-benzoquinones (e.g. 2.5-dimethyl-p-bcnzoquinone) or quinonediimides (e.g. p-phenylenediimine) are used as Class III acceptors in con- junction with dibromothymoquinone. a small but significant stimiiltation ol‘electron transport by ADP plus phosphate is Observed. However. it can be shown that this stimulation does not arise from coupling Site II but rather is due to a low rate of electron flux through coupling Site I even in the presence of dibromothymoquinone. Apparently the p-benzoquinones can catalyze an electron “bypass" around the dibro- mothymoquinone—induced block at plastoquinone. possibly by substituting partially for the natural electron carrier. If this bypass electron flow is blocked at plastocyanin by KCN treatment, the stimulation of electron transport by ADP plus phosphate is eliminated. although a high rate of phosphorylation (from Site If only) remains. Abbreviations: P/ea Iatio, the ratio of the number of molecules of ATP formed to the number of pairs ofelcctrons transported; dinietl‘quuinor‘v (DMQ). 2,5-dimcthyl-p-bcnz|oquinone. 106 158 J. M. GOULD, l). R. ORT 3. These results provide strong evidence that a profound dill'erencc exists between the two Sites of energy coupling associated with non-cyclic electron trans- port in isolated chloroplasts. That is, the rate of electron llow through coupling Site I, which is the rate-determining step in the Hill reaction. is strictly regulated by phosphorylating conditions, whereas the rate of electron llux through coupling Site ll is independent of phosphorylating conditions. 4. A model is presented which accounts for the lack of control over electron transport exhibited by coupling Site ll. It is postulated that Site ll is coupled to an essentially irreversible electron transport step. so that conditions which alfect the phosphorylation reaction would have no effect on the rate of electron transport through the coupling site. Two essentially irreversible reactions. closely associated with Photosystem ll ——the water-splitting reaction and the System ll photoac. itself— are discussed as possible locations for coupling Site ll. lNTRODL‘CTlON Saha er al.‘ were the first to point out that lipophilic Strong oxidants such as oxidized p-phenylenediamincs could intercept electrons from the chloroplast electron transport chain primarily at a point between the two photosystems. Subsequent work ltas shown that the preponderant portion of the electron transport to these “Class lll" oxidants is insensitive to the plastocyanin inltihitors KCN (ref. 2) and poly(L)-lysine (ref. 3) and the plastoquinone antagonist dibromothymoquinone“5. Thus, when electron flow to Photosystem l is blocked by one of these inhibitors, Class III acceptors are reduced by an electron pathway which includes only Photo- system ll. Recently our lttborator_v'"5 and Trebst’s laboratory“ have shown that there is a site of energy conservation closely associated with Photosystem ll-driven photo- reduction of Class III acceptors. Evidence is now accumulating that this newlydiscovered coupling site closeto Photosystem ll differsin several fundamental aspects from the well-known coupling site located after plastoquinone and before cytochrome/'7". When the two coup- ling sites are functionally isolated by partial reactions of the electron transport chain", the coupling site between plastoquinone and cytochrome [(Site I) exhibits a pH-dependent phosphorylation elliciency (Pie2 ratio) (optimal Ptt’e2=0.6 at pH 8.0—8.5) whereas the coupling site located before plastoquinone (Site ll) is less efficient, with a pH-independent P,"'e2 ratio of 0.3—0.4. In addition. we have noted‘”m that coupling Site ll apparently exerts no control over the rate of coupled electron trans- port. That is, the rate of electron flux through coupling Site ll is not stimulated by the presence of uncouplers or ADP plus phosphate (Pi). Conversely. Site I exerts tight control over the associated electron flow. responding sharply to the presence of phosphorylating or uncoupling conditions. From these results we have concluded that coupling Site I alone constitutes the rate-determining step in the reduction of conventional Hill oxidants such as ferricyanide or methylviologen". However, Trebst and Reimer“ have reported data which indicates that elec- tron transport through coupling Site ll is regulated by phosphorylating conditions. They reported that the reduction of substituted p-benmouinones in the presence of dibromothymoquinone was stimulated by the addition of A DP plus P, or amine iO7 DIFFERENCES IN CHLOROPLAST COLI’LING SITLS 15‘) uncouplers. In an effort to resolve the apparent discrepancy between their data and our own, we have rc-examined this problem in considerable detail. ln this paper we report conclusive evidence that substituted p-benzoquinones such as ”JD-dimethyl- quinone not only accept electrons at a point before the site of dibromothymoquinone inhibition5 (i.e. before plastoquinone") but also catalyze a “bypass" around the. dibromothynoquinone block. allowing electrons to pass through coupling Site I to Photosystem l. When KCN: is used to. block the bypass electron flow by inacti- vating plastocyanin”, no stimulation of electron transport by ADP plus Pi is ob- served, indicating that Sitc II in fact does not exert control over coupled electron transport. This important difference in the properties of coupling Sites land ll may reflect a fundamental dill‘ercncc iii the mode of ettcrgy transduction at the two sites. EXPERIMENTAL METHODS The techniques employed in this study were similar to those described in previous papers. Chloroplasts were isolated from fresh market sp'ttitc‘lt (.S'pi/uu'iu oleracea L.) as" described earlier". The photoreductions of S.F-dimetltquuinone and oxidized p-phenylenediamine were measured spectrophotometrically as the decrease in absorbance of the reaction mixture at 420 nm due to the reduction of excess ferricyanide'. Reactions (in a final volume of 2.0 ml) were run in thermo- stated cuvettcs at 19 ‘ C. Actinic light (>600 nm: 400 kcrgs~s"' -cm‘3) was supplied by a SOO-W slide projector and the appropiatc colored glass filters. ATP formation was determined for an aliquot of the reaction mixture as. the residual radioactivity in the aqueous phase after extracting unreacted ortho- phosphate as phosphomolybdic acid into a butanol—toluenc mixture (ltl, vv) as described by Saba and Good”. Radioactivity in the final aqueous phase was deter- mined using the Cerenkov technique of Gould t'l til”. KCN-treated chloroplasts were prepared by incubating chloroplasts in 30 m M KCN (buffered at pH 7.8) at O C for 90 min as described by Ouitrakul and Izawal. Stock solutions of 2.5-dimcthquuinmte and dibromothymoquinonc were pre- pared in ethanol-ethylene glycol tlzl. toy) and diluted so that the final concentration of organic solvent in the reaction mixture never exceeded '2"",',. RESULTS We noted previously that dibromothymoquinone. which blocks electron flow at plastoquinone. strongly inhibits electron transport and ATP formation when ferricyanide is the electron acceptor, but only partially inhibits electron transport and phosphorylation when the lipophilic Class III oxidant p-phenylenediimine (oxidized p-phenylenediamine) serves as the electron acceptor". Since the reduction ofClass lll acceptors is known to contain two components. one solely dependent on Photosystem II and one requiring both Photosystem ll and Photosystem l (ref. 2). it was concluded that dibromothymoquinone was blocking the Photosystem 1 com- ' ponent of oxidized p-phenylenediamine reduction. This conclusion was confirmed by the observation that the reduction of Class III acceptors in the presence of dibro- mothymoquinone is completely insensitixe to plastocyanin inhibition by KCN“). Fig. I shows the effect of dibromothymoquinonc on the reduction of ferri- l08 160 J. \I. COULD, D. R. ORT cyanide and oxidized p-phenylenediamine in the presence and absence of a complete phosphorylating system. As the dibromothymoquinone concentration approaches 5-10"7 M the rate of electron transport to oxidized p-phenylenediamine in the IOOCr—v r v 1' Y1 _o-a..-; v Y . I 1 C_ . ; ELECTRON rnmsponr . ‘ \ecy on2 ; i N l - l l 8 p t ‘ - soot '\ nzo_.pg°_t.u2i I (I °-5l‘°~o__._5__\_s: 3 f l '30, I\3 ; /. pl 'p l CL 1 l L 1 :s. \o / ‘ l 3 60° \3 ‘I SOCr r Y 1 v Y 1‘ O \ . ~ 1 .23 ° kl i no FORMAJICN t‘. l l r: t I ' wt ‘0' 400i u o—.r 4 —. soot- E \ 2 CCy l .0 '\. .1. EC . _p . 5‘ \ poo. . > i \/ i \ f — .p i K: ' .\ l 3 2003\(1. . 1 ix zoo» ._-. “i I .\\ / | -n l .\ rec l l \J ' V l \‘O—Oll .3 ; . l I: O L L 1 L 4 ' 3 o l J_ 1. .__.;l 0 l 2 3 4 5 0 l : 3 ‘ 5 onenouornmooumoss tmio’) Fig. I. Effect of the plastoquinone-antagonist dibromothymoquinone on electron transport and ATP formation associated with the photoreduction of ferricyanide and oxidized p-phcnylene- diamine by isolated chloroplasts. The reacuon mixture contained (M \1 sucrose. ‘ mM Metlg, 50 mM Tricine-NaOH buffer (pr 8.0), 0.75 mM ADP, 5 m M Nagll‘l'JI’O.‘ (when added), chloro- plasts equivalent to IS ug chlorophyll, and the indicated acceptor system. The acceptor systems were: Fecy, 0.4 mM potassium ferricyanide; Pl)“. 0.5 mm p-phenylencdiaminc plus 1.5 mM potassium ferricyanide. Note that as the Photosystem I component of PD... reduction is elimi- nated. the stimulation of electron transport by Pi is also eliminated. even though a high rate of ATP formation remains. Also note that at dibromothyinoquinone concentrations 3 2.5- to “7 \f, where dibromothymoquinone itself functions as a Class lll acceptori”. the stimulation of electron transport by P. is not observed. although phosphorylation. with the charactcrzstic Site If P c: ratio of 0.3—0.4, does occur. phosphorylating (+Pi) system falls to the level of the nonphosphorylating (—P,) system. Nevertheless, this dibromothymoquinone-insensitive electron transport remains firmly coupled to ATP formation, even though the stimulation of electron transport by ADP plus Pi is no longer observed. Similar results can be seen when ferricyanide acts as the electron acceptor. This is because dibromothymoquinone. at concentrations greater than 2.5- If)‘7 M. not only blocks at plastoquinone but also functions as a Class III electron acceptor”), the reduced dibromothymoquinone being rapidly reoxidized by the excess ferricyanide present in the reaction mixture. In both systems the P/ez ratio falls to around 0.4. the characteristic etliciency of coupling Site II2'4"5'°"°. Since both systems (in the presence of 5- lO’7 \1 dibromo- thymoquinone) are insensitive to plastocyanin inhibition by KCN, we can conclude that only coupling Site If is involved. While the data in Fig. 1 clearly show that coupling Site [I exerts no control over Photosystem II electron transport, Trebst and Reimer“, using substituted p-benzoquinones as Class III acceptors, have shown that electron transport to these compounds is stimulated by ADP plus Pi and by amine uncoupling, even in the pres- ence of dibromothymoquinone concentrations which completely block electron transport to Photosystem I. From this data they concluded that coupling Site If does exert control over the rate ofelectron transport. To resolve this apparent discrepancy lO9 Dll-‘FERlZNClJi IN CHIEFIUVH’LAST COUPLING Sll‘l-‘S lol with our own results. we perl‘ormed similar experiments using 2.5-diinetli3l-p- benzoquinone as the Class lll acceptor. Fig. 2A shows that in the mixed system (i.e. tllttlL‘lllquultlt‘llC reduction by both Photosystem ll alone and Photosystem ll ,a/ux Photosystem l), considerable stimulation ol‘ electron transport by the complete phosphorslation s_\.st"m (+l’.) is observed. These data contirm the earlier t“..‘\ttlts ol'Saha VIN/.1. ll‘ 5- l0": .\l dihro- mothymoquinone is added (Fig. 213i. howeser, the rate ol‘ electron transport is inhibited, indicating that the Plh.)lt)5)slc‘llt l component ol‘ dinietliyltiuinone reduc- tionz°5 has been largely eliminated. When no .llll‘t‘ll‘.}‘lL]t!lllUllc‘ is present in the reaction mixture (dimethsluuinone=0. Fig. am. the residual ittte ol' electron trans- port. which is due to the Class Ill-type reduction of l'eri'icsaaitle i'i'a dibromothymo- quinonc'o, shows no stimulation by ADP p/us' Pi. \ex'ertl‘eless. when dintetlnl- quinone is added. a small but signilicant stimulation of e.eciron transport by ADP plus Pi is observed. This conlirms the lindings ol' Trebst and Reimer" that control (——V———T.—‘"l"“’Y—1 l T‘—'—"F' .-,-.'_ r-‘"v—i —v v j '0" A .J ‘OL 8 ii ii)-- C 1 l‘\ l ’ I i 1 a ‘ m ‘ u? 05‘»- \° ’ “1' 3" w-v— 1‘ 3 0 st- l [g l | a p-D—'~——-1v~-—--———-‘-i :1 i-n—.——p-————,—i ' ‘ I ol 1— L A g} ("i 4 L _.__t_....' 0i L L L A _A 20901’ T W v f v 'O’J‘)"“'”‘r -" 'V‘“ _Y ' " —Y‘—fi °OOO{ 7‘ Y 7 v . u i I l I 1 b -.n r. ——'_-_—_._ L t .9. fl. ' t . ‘ s toot /,. . . ., . _/ _s r. 1 ° \ ’ ~’ ' _ / op . I h. :0 i Q E ' l ’ ’ l ,, E Y 3 o ,. l . - . I I n- “ ' ‘ ' \ A -9 . _I,__—- -—< L iZOOl- '/ / ‘ 4 v p / ‘ p " (10A.- 4/A _ . C .I l" W‘)-' ~ / I g / / l a N. l g‘ . / n ' l L I L i T: f, -l .i “ 400 " l 800 n w’)’)- ,2 - w t- . .. 3 . ATP d 1 {I 1 T" ! 'i l \ ' i. in. g 1 ir' 1 ’ s Q-. ° A- A s. (., 4 - _‘0 4 U 600 . l c a r ATP . e ‘ “Y9 I‘. i . f, I rcnnicvmot acouf s I ‘,,_-~.-————--"”‘ 7 i . __ _' U l/ l n u / I U a") U U / 1’ 1 9L 1 A A 1 ‘- k . -._._._t.._-_. - __‘.__.___._. ; 3--.--..i i ' 4 3 C35 05 C75 ’J 4 3“! U“ '1" .; 3 325 J‘.‘ 775 .C owr YiiLOIJiNONE (mm Dis" 'n‘ We lemon: “.in .1 w; T-Wt Du NONE .mw Fig. 2. ElTect of the electron transport inhibitors Llll‘rt‘lllt‘lll)ll‘thttlllt‘ttc and KCN on electron transport (E.T.) and phosphor)lation tA'l'l’) \shcn diincth}ltitiinone is the electron acceptor. The basic reaction mixture is as descrthed in lie. I when t'ci'z'icsanide “as the electron acceptor. Specific conditions are as follows: (A) .\'o inhibitor added. \‘ote 'hat \sll'c‘t‘. no dimeth}ltiuinone is present (i.e. ferricyanide is being reduced via the normal Hzll reaction) there is a large stimula- tion of electron transport by the coinpiete phtisphorslataig system t —- Pi). \\ hen dimethqueznonc is added, a large increase in the rates of electron transport is observed. although the absolute amount of stimulation by Pi remains about the same. (ll) Dihromothymotniinone (S- 10”7 M) was added. Here rates of electron transport are losser since dibromothyinoutiinone blocks the Photosystem l component of dimethquuinone reduction. Note that in the absence of dinieths-l- quinone (i.e. when dibroniothymoqtiinone serses as the electron acceptor) no stimulation by [\Dl’p/us Pi is obsers ed. \\ hen increasing concentrations ol‘ dimet'iquuinoiic are added, hoxses'cr. a small but significant stimulation of electron transport by." the complete ( -~ l‘;l system is observed. (C) Chloroplasts blocked at plastocyanin by K(‘.\' treatment (see Methods): 5° 10 7 .\l dibromo- thymoquinone added. \‘ote that the stimulation of electron transport h} the complete phosphory- lating system (.see ll) is aholished by Plit\lUc‘_\;‘.nlll inhibition. indicataie that electrons are by- Passing the dibromoth}itioqtiinone--iiduced 'nlocls. Nesertheles‘s. a substantial rate of ATP for- mation remains even when this by pass is blocked by KCN. NO 162 J. M. GOULD, D. R. ()RT is present in this system, even in the presence of a dibi'oinotliyinottuinone block. However. by using the plastocyanin inhibitor KC\ it is possible to show that this control ol' electron transport by phosphorylating ‘oziditions is not due to coupling Site ll. ll'electron flow is completely blocked at plastoeuinone by dibromo- thymoquinone, then inactivation of plastocyanin by KCN should have no el‘i'ect on dimethquuinone reduction. However, as Fig. 2C shots s. treatmen: ol' chloroplasts with KCN completely eliminates the stimulation ot' electron transport by ADP plus Pi seen iii Fig. QB. Nevertheless, tte remaining electron transport is coupled to ATP formation with an ellleieney of 0.3—0.4. Thus. it can be concluded that in the presence ol'dibromothymoquinone plus dimethyltitiinoite. there is a small amount of electron leakage around the dihrornothyntotitiistone block \siiieli allows a slow rate of electron tlux through coupling Site l. Since Site l exerts tight control over electron transport". this would account for the stimulation ol‘ electron how by ADl’ plus Pi which is observed in this system. lndeed. when this electron leakage is blocked at plastocyanin. the remaining pure Photosystem ll reaction. utilizing only coupling Site H, is not stimulated by ADP plus Pi. it should be pointed otit that we have not observed this leakage phenomenon when oxidized p-plien)lenediamine tl-"ig. l) r . 1 T] '°ll. . more—e"? — ._.-j_j l \r" ‘CC' l A | \\ l T: \ ‘3' 0 5i. “1: ’ pom (oOBMIBl -l f , \ H35 - p.09). Q ‘l‘g\’\/ r? I \O‘ 1 \.'.§(\\ . l (_j | x.\ . __ _ - Z. , L] I \ '200. —‘v— r i E 1500.- "l T‘ l‘ I \ i ‘ r \n l \,PD°, toes-mi \7 _ , ‘ 3 -\ ; tOOO U -i P \ ‘ z T \ / T'\ \ g l ”s l) ‘\ 6' ' "' l :‘ t, F- ..- ___ ‘ I L ’ J m lOnO" -/-/ . ‘\.\ U 800l- .\ (' ./' 1° \ (L / L s' O- 9} l / , / a E ‘5 :2 Goal- /\‘." 'l E ' /. ”3C'-—>"em 2; l l - x m ,1 z) ‘ .’ 3 i C: ‘ i .I... I ‘E’ ‘W q C JCO -4| 3 l U ‘ . 6 l .i . l ld D I 3 200‘ ATP 'J GL 1‘ A . l Os-ML__.__+ .__.. ._.....______._,_._ o :. no .3 7 b if- is 2:) METHYLAMIHE (MM) METHYLAV'VC ”7".” Fig. 3. ElTect ol' the uncoupler methylamine hydrochloride on electron transport (H.T.) and ATI’ formation when ferricyanide (Fecy) and oxidi'led p-p‘nenylenediamiiie (l’DF‘) serve as electron ac- ceptors. Reaction conditions are as in Fig. I. When I’D.“ \L‘l'\Cd as the electron acceptor, 5- it) ‘7 \l dibromothymoquinone (DBMIB) was added to block the Photosystem l component of PD.“ reduction. Note that the ferricyanide system shows a big stimulation ot‘ electron tiansport as the rate limitation at coupling Site I is relieved, white electron transport in the Pl)... ssstc‘m is in. hibited us Site ll becomes uncoupled. Fig. 4. ElTeet of methylamine on the rate of electron transport a hen ferricyanide (Fecy) and oxidized p~phenylenediamine (Pl)..,‘) are the electron acceptors. Reaction conditions are as in Fig. 3 except that dibromothyinoquinone was omitted from the PD.“ system to climi'iatc a secoii~ dtiry ell'ect of the inhibitor on the quantum elliciency ol' l’l‘."lt‘\‘}s‘lc‘t“ ll 't'et'. ltli. lll l)lFFERENCES lN CHLOROPLAST COL’PLlNG SlTlES 163 or other, substituted quinonediimides are used as Class lll acceptors, suggesting that the p-benzoquinones may be able to substitute partially for the natural electron carrier plastoquinone. The effect ol‘ the uncoupler methylamine on electron transport and phos- phorylation associated with the reduction of ferricyanide and oxidized p-phenyl- enediamine (in the presence of 5- 10‘7 .\l dibromothymoquinone) is shown in Fig. 3. As is already widely known, methylamine stimulates the rate ol‘ electron transport when ferricyanide is the electron acceptor by uncoupling electron llow from a rate- limiting energy conservation reaction”. Since we have shown previously that coup- ling Site l is the rate-dctermining step for the Hill rcacuon", we can coneliide that methylamine‘s preponderant ell'ect on electron transport is by releasing the rate- limitation at Site I. Methylamine has an entirely ditlercnt ell'eCI on a Photosystem ll partial reaction (utilizing only Site ll). however. Instead ot‘ stimulating the rate of electron transport as Site ll becomes uncoupled. methylamine inhibits electron llow. Since this inhibition increases with increasing concentrations of methylamine. even after ATP formation has been completely abolished. it seems likely that the inhibi- tion is actually due-to a secondary elTect ol' the amine on Photosystem 'lI. lndeed. high concentrations of methylamine htve been shown to inhibit the water-splitting reaction”. In this experiment (Fig. 3l—tlie rate of electron transport iii the presence of >5 mM methylamine is somewhat lower when oxidized p-phciiylenedlamine is the electron acceptor than when l'errieyanide is the electron acceptor. This is due to the dibromothymoquinonc present iii the p-phcnylenediamine system, since dibromo- thymoquinone has been shown to have a secondary etl'ect on the quantum etliciency ol‘ Photosystem ”'0. In the absence of dibromothymoquinone (Fig. 4) similar results are obtained for the clTect ol' methylamine on ferricyanide and oxidiied ,‘i-phenylene- diamine reduction. It is clear that. as the rate limitation at coupling Site | is released, the rate of ferricyanide reduction increases until the secondary inhibition of Photo- system ” by methylamine becomes the rate-limiting l‘actor. DlSCUSSlON There is an impressive amount ot‘ evidence accumulating which indicates that the mechanisms of energy conservation at Sites 1 and ll are not identical. When these sites are isolated by partial reactions ol‘ the electron transport chain". it can be demonstrated that they ditl'er in their coupling elliciencies tP'e2 ratios) and in the elTect ol‘ pH on these etliciencies. Site ll exhibits a characteristic Pie: ratio of 0.3—0.4 which is practically pH-independent from pH 6 to 9. whereas Site I. which is the rate-limiting step for the Hill reaction. exhibits a strongly pl-l-dcpendent Pie2 ratio having an optimal value ofabout 0.6 at pH 3.0—8.5. Furthermore, it has recently been shown that HgClz, which is an energy transl'cr inhibitor in chloroplasts”, can preferentially inhibit the coupling reaction at Site I without till'ecting ATP for- mation supported by Site ”‘3. In this paper we have reconfirmed that, unlike Site I, coupling Site It does not exert control over the rate ol' associated electron this. That is, the rate ol'electron transport through coupling Site ll is independent of the presence ol‘ ADP plus Pi or uncouplers. Nevertheless, under phosphorylating conditions ATP l'ormation supported by Site ll can occur at very high rates. ll2 lti-l .l. M. GOULD, l). R. ORT Furthermore, we have presented data which allows a reinterpretation of the findings of Trebst and Reimer“. who concluded from experiments with dibromo- thymoquinone and 2,6-dimethyl-f-bct‘t7oqttinonc that coupling Site ll docs exert control over electron transport. Apparently certain p-benzottuinones. in the presence of dibromothymoquinone. can cataly7c an electron bypass around the dibromo- thymoquinone-induced plastoquinone block. perhaps by substituting for plastoqui- none.This does not seem unlikely in view of the structural similarities between these p-benzoquinones and plastoquinone. When KCN blocks this bypass. howexer. no control over electron transport by Site It is observed. lndeed. Trebst and Reimer“ noted that when they used highcr concentrations of LlllN'Ulllt‘lil)l‘l(‘kllllll(lllc an the presence offcrricyanide (so that dtbromothymouuinone itself functioned as a Class lll acceptor) no ell'ect of uncouplers on electron [ritltspttt't was observed. This obser- vation is in agreement with ottr own results obtained in a more extensive study of dibromothymoquinone as an electron acceptor'”. lt is also important to note that oxidized p-phenylcncdiamines. when used as Class lll acceptors. do not cataly7e this bypass reaction around the dibromothymoquinone block. It is possible to construct a model which explains the observed differences between Site H and Site I in their ability to regulate electron transport. In many respects coupling Site I resembles sites associated with .the mitochondrial respiratory chain°. By analogy with mitochondria”, the electron transport between adjacent electron carriers (A and l3) at Site l can be viewed as an equilibrium system. Thus. in the light, when Photosystem l is rapidly draining electrons from ll. the reaction (/\«>»B) would be pulled to the right. However. as the high energy state (~ ) generated in the coupled energy conservation reaction begins to accumulate. a back pressure is created against the flow ofelcctrons from A to B by reversal of the energy conser- vation steps. When a complete phosphorylating system (i.e. ADI’ p/us P.) is present, however, the pool of high energy intermediate is much smaller since it is continually being utilized to drive ATP Formation. Thus the back pressure exerted by this pool is diminished and electron transport from A to B is stimulated. Smtilarly, in the pres- ence of uncouplers, the high energy state is rapidly dissipated and no signilicant back pressure is present. allowing very high rates ofelectron transport. Coupling Site ll does not exhibit the tight control mer electron transport seen at Site I. This can be readily explained. however. if the oxidation reduction reaction which gives rise to the energy conservation step at Site ll is essentially an irreversible step. That is. the nature of the forward reaction (A~ ll) is such that the reverse reaction is thermodynamically prevented. In this case. the accumulation of the high energy intermediate or state (~) would still exert a back p"essure on the forward reaction, bttt this back pressure would have no etl‘ect on tftc rate of electron transport from A to B. This would account for the fact that conditions which drain or dissipate the high energy intermediate pool do not etl‘cct the rate ofelectron llow at Site It. Several observations lead us to believe that this model does indeed provide a reasonable explanation for the dillcrcnccs between Site ll and Site l discussed in this paper. We have shown previously that coupling Site ll occurs at a point in the electron transport chain before the electrons from the independent Photosystem ll units are pooled"). This indicates that coupling Site It is located prior to plasto- quinone in the electron transport chain. The insensitivity of the phosphorylation ll3 DIFFERENCES IN CHLOROPLAST COUPLING SITES 165 associated with the reduction of Class III acceptors to the plastoquinone antagonist dibromothymoquinone lends strong support to this argument. lndeed. :he existence of a Photosystem ll-driven “proton pump" before plastoquinone has beett demon- strated". There are at least two reactions in this portion of the electron transport chain which could be considered essentially irreversible. One ofthese is the System ll photo- act itself. It has been suggested that a photochemical quantum conversion results in the formation of a reduced acceptor Q‘ and an midi/ed donor Z+ on opposite sides of the thylakoid membrane (e.g. ref. 20). According to this model the resulting electrical lield or membrane potential could serve as an energy reservoir to drive ATP formation as elaborated by Mitchell“. A second essentially irreversible reaction closely associated with Photosystem ll eleCtron transport is the water-splitting reaction. The existence of a coupling site on the water-oxidi7ing side of Photosystem ll has already been considered by several autliors"'23‘2“. It has been suggested that the protons from water oxidation are released to the inside of the thylakoid membrane. generating a transmembrane H+ gradient which is capable of driving ATP formation”. Experiments are currently in progress in an attempt to further define the exact location of coupling Site H in chloroplasts. ACKNOWLEDG EM ENTS The authors would like to thank Drs S. lzawa and \l. E. Good for many helpful comments on this work. We would also like to acknowledge valuable dis- cussions with Dr Bessel Kok concerning the interpretation of the control mechanisms discussed above. This work was supported by grants (GB 32657 and GB 37959.\’) from the National Science Foundation. U.S./\. REFERENCES l Saha. S., Ouitrakul, R., Izawa. S. and Good. N. E. (1971) J. Biol. Chem. 246, 3204-3200 2 Ouitrakul, R. and Izawa. 5. (NH) [Jim-Iii)”. ”flip/LVN. .‘Jt'ftl 305. lOS—l IX 3 Urt, D. R., Izawa, S., Good. N. E. and Krogmann. D. W. (W73) FIE/{S Len. 3|, 119422 4 Could, J. M.. Izawa. S. and Good. N. E. (1973) Fed. Proc. 32, ('32 S Izawa. S., Gould. J. Vl.. Ort, D. R., Felker. l’. and Good. I '. E. (1973) Rim/rim. Biop/ii's. .ilcra 305. ll9-128 6 Trebst. A. and Reimer. S. “973) Biochim. Biophys. Arm 305, 139—139 7 Avron, M. and Chance. B. (Who) [frank/lawn Symp. b'l‘ul. l9, l49~l"0 8 Bohme, H. and Cramer, \V. .-\. (W73) Iii'or'r'iwni.slr_i' l l, l IFS l ”‘0 9 (iould, J. M. and Izawa. S. (l‘r’Bl Biochim. [hop/Li's. {it (a 3M. Zl l—223 10 Could, J. M. and Dawn. S. (W73) lz'ur. J. ”inc/mm. 37. INS- I”: ll llbhmc, H., Reimer. S. and Trebst, A. (W72) Z. Naturforsch. 30b. 34l 4‘53 12 Izawa. S., Kraayenhof, R., Ruuge, E. K. and DeVault. D. (1973) Biochim. Biophys. Acta 3H. 328—339 13 Saha. S. and Good, N. E. ”9703 J. Biol. (Item. 245. 50l7~502l l4 Gould, J. M., Cather, R. and Winget, G. D. (1972) .‘llllll. Dior/win. 50. $40-$48 15 Good. N. E. (I960) Biochim. Biophys. Arm 4”. 502 —5l7 lo Izawa. 5.. Heath, R. L. and Hind. (i. (l‘Nv‘l‘ lz'im/u'm. ”hip/’02". Arm ISO, SSS-398 l7 Izawa, S. and Good. N. E. (WIN) I’I'Ut'. I’I'Iu/man/I. Rm. 3. llb‘b'»! :93 IS Bradeen. D. A.. Gould. J. NIL. ()i t. D. R. and Winget. (3. f). (HTS) I’m/ii Physiol., in the press H4 166 J. \l. COULD, I). R. (\RT 19 Chance. ll. (l~)'72) l-‘lfi'i'S Luz. 23, 2-30 :0 \Vitt. H. T. (l‘)7l) Q. /\'t't'. [hop/It's. 4, 365—477 2! Mitchell, P. (196m Rial. RL'V. 41,4454“): :2 Schwartz, M. (1963') .Vulurt' Zl‘), ()lS-‘Jl‘.’ 23 Yamashita, T. and Butler, \\. l.-. (tans; Hun! l’lrrw'ul. 43, l‘)7H-l‘)\‘(i 24 Bdltmc, H. and Trebst. A. (l‘)(\‘)) Bind/rm. [hop/its sic/u lbi‘. l37- l‘i‘i 25 Junge, W., Rumberg, ll. and Scltrotler, l-l. (l‘f'tl) lim‘. J. {inn/1cm. l-l 575—53! PLEASE NOTE: Page 115 seems to be missing in numbering only as text follows. UNIVERSITY MICROFILMS INTERNATIONAL APPENDIX V SITE~SPECIFIC INHIBITION OF PHOTOPHOSPHORYLATION IN ISOLATED SPINACH CHLOROPLASTS BY MERCURIC CHLORIDE 117 Plant Physiol. (1973) 52, 680—682 Site-specific Inhibition of Photophosphorylation in Isolated Spinach Chlor0plasts by Mercuric Chloride‘ DAVID A. Bunsen AND G. DOUGLAS Wmoer Received for publication June 4, 1973 Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221 J. MICHAEL GOULD AND DONALD R. Oar Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824 ABSTRACT Photophosphorylation associated with noncyclic electron transport in isolated spinach (Spinach oleracea) chloroplasts is inhibited to approximately 50% by low concentrations of HgCh (less than 1 mole Hg”/mg chlorophyll) when the elec- tron transport pathway includes both sites of energy coupling. Reactions involving only a part of the electron transport system can give a functional isolation oi at least two sites coupled to phosphorylation. Only one of these sites, located between the oxidation of plastoquinone and the reduction of cytochrome I, is sensitive to mercuric chloride. The energy conservation site located before plastoquinone and close to photosystem ll is unafl'ected by HgCl. concentrations up to Ill-fold those re- quired to inhibit phosphorylation by the coupling site after plastoquinone. This site-specific inhibition may reflect a mechanistic diflerence in the mode of energy coupling at the two coupling sites or a variable accessibility of HgCl. to these sites. Concentrations of Hng, which inhibit steady state phos- phorylation, do not inhibit dark phosphorylation after illumi- nation (Xs). suggesting that HgCl. aflects a step in the cou- pling mechanism prior to the terminal step of ATP formation. Recent data from several laboratories indicate that there are at least two sites of energy coupling associated with noncyclic electron transport in isolated chloroplasts (5-7, 11, 17-20). By utilizing various electron donor-acceptor systems in con- junction with several new inhibitors of electron transport, these energy-coupling sites can be functionally isolated and charac- terized. One site closely associated with photosystem II (5), differs in several respects from a second, well recognized site coupled to electron transport from plastoquinone to cyto- chrome /. (3). The photosystem Il-dependent energy coupling site exhibits no control over coupled electron transport and has a P/e.‘ ratio of about 0.4 (5-7, 17. 18). Furthermore, this We. 1J.M.G. and D.R.O. were supported by Grants GB 22657 and GB 37959X from the National Science Foundation. ‘ Abbreviations: Ne. ratio: the ratio of the number of molecules of ATP formed to the number of pairs of electrons transported; DCIPH; reduced 2.6-dichlorophenolindophenol; HEPPS: hydroxy- ratio is practically pH independent (5, 6). On the other hand, the coupling site between plastoquinone and cytochrome I. which is the rate-limiting step for the Hill reaction, exhibits control over electron transport and has a pH-dependent P/e. ratio of about 0.6 (pi-l 8.0—8.5) (6). Because of the apparent dif- ferences in the characteristics of the two coupling sites, it was of interest to study the efiect of specific inhibitors of the phos- phorylation reaction (energy transfer inhibitors) on each cou- pling site. In this communication, we report that the energy transfer inhibitor HgCl, (l I) specifically inhibits ATP forma- tion supported by the coupling site between plastoquinone and cytochrome I while net afiecting ATP formation supported by the coupling site close to photosystem II. MATERIAIS AND METHODS Spinach (Spinacia oleracea) chloroplasts were prepared as described previously (21). except TES-NaOi-l bufier replaced Tricine in the isolation media, since Tricine strongly binds Hg. Reactions were run in a thermostatted vessel at 19 C using strong light (>400 kerg/cm3-sec). In all cases, the chloroplasts were incubated with HgCl. for 30 sec before the addition of donors. acceptors, or inhibitors. Electron transport using oxidized p-phenylencdiamines, sub- stituted p-benzoquinones, or ferricyanide as the electron ace ceptor was followed spectmphotometrically, as described else- where (19). MV reduction was measured as oxygen uptake (10) with a Clark-type membrane-covered electrode. ATP forma- tion was assayed using a modified procedure of Avron (2). Ra- dioactivity was measured using the Cerenkov technique of Gould et al. (4). RESULTS AND DISCUSSION Figure 1 shows the efiect of low concentrations of HgCl. on electron transport and phosphorylation using three difierent electron donor-acceptor systems. As previously reported (11). the over-all Hill reaction with ferricyanide (Fig. 1A) or MV (Table I) as the electron acceptor is inhibited in direct propor- tion to the added HgCl. up to approximately 35 to 40 nmoles of HgCl./mg Chl. Although very high concentrations of HgCl. (greater than 1 mole/ mg Chi) result in nonspecific electron ethylpiperazinepropsnesulfonic acid; FeCy: potassium ferricyanide; DBMIB: dibromothymoquinone: DMQ: 2.5-dimethyl-p-benzoqui- none; PD..: oxidized p-phenylenediamine; DAD“: oxidized dia- minodurene; MV: methylviologen. 118 INHIBITION OF PHOTOPHOSPHORYLATION Plant Physiol. Vol. 52, 1973 A) Mac - FsCy a) ocwnz-mv a) mac - so" '4 r E Y («anti ‘1 50°F: '- 6‘6 \0‘ 1" '3 atria!” “ ' r 400C 1)- :, x fines-mt) a“- .I if a 300 s)- (T (COMET!) 1r- -( 'A. x trivalent) u (couture) 200 \ at t- D.) U. t-Pu) art- ' ‘W‘A Q 'm Wu--. ----- J- ‘p q \ Electron Transport or ATP Formation l l l 0.2 0.4 0.2 0.4 0.2 0.4 HgCl: (alleles/mg Chlorophyll) FIG. I. Efiect of HgCl: on electron transport (E.T.) and phos- phorylation associated with various electron transport pathways. The reaction mixture (2 ml) contained 50 mM HEPPS-NaOH buffer (pl-I 8.2). 2 mM MgCl:. 0.1 M sucrose. 1 mM ADP. 5 mM NaJinPOH chloroplasts containing 40 pg of chlorophyll. and the indicated donor-acceptor system. These systems were: A. 0.4 mM ferricyanide; B, 0.4 mM DCIPI-h. 2.5 mM L-ascorbate and 50 “M MV: C. 0.5 mM p-phenylenediamine (PD) plus 1.4 mat ferricyanide. When added. methylamine was 10 mM. In the DCIPH: -° MV system (B). 1 an DCMU was added to block electron transport from photosystem II. When PD... was the electron acceptor (C). 0.5 uM DBMIB was added to block the photosystem I component of PD... reduction (1). Note that only the H.-O -r PD... system, which does not utilize the rate-determining coupling site after plastoquinone, is insensitive to inhibition by HgClg. Rates of electron transport and ATP forma- tion are given in umoles/hr-mg chlorophyll. Table I. Eflect of HgCl, on Photophosphorylation in Spinach C hloroplasts with Various Electron Acceptor: The reaction mixture (3 ml) contained 20 mM HEPPS-NaOH buffer (pH 8.2). 50 mM sucrose. 1 mM MgCl . 1 mM ADP, 5 mM Na, H“PO.. chloroplasts containing 60 pg of Chl, and the indicated electron acceptor system. These systems were: I: 50 pH MV; 2: 10 an DBMIB plus 0.4 mM ferricyanide; 3: 0.5 mM DMQ plus 0.4 mM ferricyanide; 4: 0.5 mm DAD plus 1.4 mM ferricyanide. A low concentration of dibromothymoquinone (0.5 an) was added to reactions 3 and 4 to inhibit the photosystem I component of DA D.,. and DMQ reduction (2). Rates are given in nmoles ATP’hr-mg Chl. Note that only the H,O .. MV system is sensitive to HgCl,. : 1 Phosphorylation Rate 1 Exocriment f Electron Acceptor! Microtnoles HgCl: added/mg Chl None I 005 i 0.25 .\'o. ' , nmoles ml 71’ 'hr-mg Chl 1 MV _ 200 I 102 " 105 2 DBMIB 47 46 42 3 DM Q . 71 65 64 4 DAD... 217 200 195 transport inhibition (1 l, 13), the low levels of HgCl, used here (less than 1 mole/ mg Chl) inhibit ATP formation (and that portion of the electron transport dependent upon phosphoryla- tion) to a plateau of approximately 50%. Contrary to the re- sults of Miles et al. (16), however, we find the same degree of inhibition by HgCl, when electron transport is measured spec- trophotometrically or as oxygen evolution. Neither basal (-Pi) or uncoupled (+methylamine) electron transport is significantly affected by HgC1.. indicating that HgCl. does act as an energy- transfer inhibitor rather than an electron-transport inhibitor at these concentrations (1 l). Chloroplasts which have been un- 681 coupled by EDTA treatment, which removes CF. (1), are also insensitive to HgC1,. DCIPI-I3 (in the presence of DCMU and ascorbate) donates electrons at a point before the rate-limiting coupling site on the electron transport chain (i.e. before cytochrome /) (6, 9, 15). It has recently been shown that the photosystem I-depend- ent partial reaction DCIPH, -r MV includes only the rate- limiting coupling site after plastoquinone and not the coupling site before plastoquinone (6). Figure 18 shows that HgCl, af- fects the partial reaction DCIPH2 -> MV and the over-all reac- tion H.,.O -> FeCy similarly, indicating that both pathways in- clude the HgClg-sensitive site. Moreover. since this coupling site constitutes the primary rate-limiting step for both electron transport reactions, :1 similar 50% sensitivity to HgCl, should be observed for both systems (compare Fig. l, A and B). However, when electrons from water reduce lipophilic ac— ceptors (class III acceptors [19]). such as oxidized p-phenyl- enediamine (PD...), a different effect of HgCl is observed. These lipophilic oxidants (in the presence of the plastocyanin inhibitors KCN or poly-L-lysine (17) or the plastoquinone an- tagonist dibromothymoquinone (12. 20)) accept electrons at a point in the electron transport chain before plastoquinone (5). Thus the partial reaction H,O -r PD... includes the photosystem II-dependent phosphorylation site. but not the rate-determin- ing site after plastoquinone. As Figure 1C shows, electron transport and phosphorylation associated with the partial reac- tion H.O -r PD... is completely insensitive to HgCl... inhibition. Several other class III acceptors were also tested for HgCl, inhibition with similar results (Table I). The absence of inhibition by HgCl. with class III electron acceptors is probably not a result of fortuitous reaction condi- tions that mask the inhibition. In all cases, chloroplasts were incubated with HgCl. for 30 sec before the addition of the ac- ceptor system. Since SH-compounds can reverse HgCl, inhibi- tion ( l 1). it seems likely that Hg“ is reacting with a membrane sulfhydryl group. Thus it is unlikely that binding between Hg” Table II. Efl'cct of HgCl, on Pastillumination ATP Formation (X1) HgCh. triphenyltin chloride. or methylamine (at the final con- centrations indicated) were present in the dark. phosphorylation stage only. ChlorOplasts containing 100 mg of Chl were illuminated with white light (>400 kergs/cmlseC) for 20 sec in a continuously Stirred reaction mixture (2 ml) containing 0.1 M sucrose. 50 mm NaCl. 2 mM MgCl,. IO mM MES-NaOH buffer (pH 6.0), and 5 an pyocyanine. Immediately after shutting off the light. 0.25 ml of a stock solution containing the test compound was added to the reaction mixture with a syringe. After 5 sec 0.75 ml of a strongly buti'ered ADP phosphate mixture (0.!5 M HEPPS-NaOH buffer (pl-i 8.0), 3 mM ADP. 15 mM Na,H"PO.) was quickly injected to initiate ATP formation. After 20 see the dark phosphorylation was terminated by addition of 0.5 m1 of I N perchloric acid. All reactions were run in a thermostatted cuvette at 19 C. Note that HgCl, did not significantly affect the yield of X: at concentrations which strongly inhibit steady state phosphorylation. The tributyl- tin analog triphenyltin, which is a potent energy transfer inhibitor in chloroplasts (unpublished observations of J.M.G.), docs abolish X5. however. as does the uncoupler methylamine. Addition (dark stage) ATP Formed Inhibition amulet/mg Chl : % *— None 64 { HgCl, (20 nmoles/mg Chl) 58 | 9.5 HgCl, (200 nmoles/mg Chl) 52 l 18 Triphenyltin chloride (10 uM) 8.8 i 87 Methylamine hydrochloride (5 mm 12 ' 81 119 682 and the quinonediimides or substituted benzoquinones used here as class III acceptors would be strong enough to reverse the inhibition. Furthermore, UV spectra of DMQ, DAD... PD... and DBMIB remained virtually unchanged in the pres- ence of high concentrations (33 nM) of HgCl.. again suggesting that little or no binding is occurring. This evidence suggests that HgCl. is not interacting chemically with the class III ac- ceptors used here and supports our contention that the energy- coupling site close to photosystem II is insensitive to HgCl.. Table 11 presents evidence that HgCl, acts on an early step of energy conservation rather than a terminal reaction of ATP formation. Concentrations of HgCl.. which strongly inhibit steady state phosphorylation (e.g. 11.0 -r MV), were demon- strated to have virtually no effect on the chloroplast’s ability to synthesize ATP in the dark after brief illumination (Xe) (8). However. methylamine, an uncoupler. and triphenyltin chlo- ride, an energy transfer inhibitor which acts on a terminal re- action of ATP formation. decrease the yield of X. significantly (8: unpublished observations of J.M.G.). The idea that HgCl. affects an early stage of energy transfer is also supported by the fact that the trypsin activated ATPase activity of CF. and whole chloroplasts is not afiected by the low levels of HgCl. used here (data not shown). The results presented herein lend strong support to the argu- ment that the two known sites of energy coupling associated with noncyclic electron flow in chloroplasts may exhibit mech- anistic differences in their mode of energy transfer (5. 6). It has previously been shown that these coupling sites differ in their response to pH (5, 6), uncouplers, ADP + P. (7). and in phos- phorylation efficiency (P/ e. ratio) (6). In addition. we have now demonstrated that the energy-transfer inhibitor HgCl. is spe- cific for the rate-determining coupling site after plastoquinone and before cytochrome /. This selectivity may be due to a vari- able accessibility of HgCl. to the coupling sites. or. alterna- tively. to a basic difference in the mechanism of the early steps of energy conservation at the two sites. A discussion of why the HgCl. sensitive coupling site is only partially sensitive (50%) to the inhibitor is beyond the scope of this report. In a subsequent publication, a more detailed study of HgCl, inhibition of chloroplast reactions will be presented, with emphasis on the nature of HgCl. inhibition and the signifi- cance of the 50% inhibition plateau. Acknowledgments—The authors wish to thank S. Izawa for many valuable sug- gesttons. WWII CITID f. Avron'. M. 1903. A coupling factor in photophosphorylation. Biochim. Biophys. Acta 77: 099-700. 2. Afloat. M. 1900. Photophosphorylation by swiss chard chloroplasts. Biochim. Biophys. Acta i0: 251-265. BRADEEN, GOULD, ORT, AND WINGET Plant Physiol. Vol. 52, 1973 3. Binnie. H. .\.\'D W. A. CRAMER. 1912. Locallltlllull HI .1 me 01 t-netgy coupling between plastoquinone and cytochrome I In the electron transport. chain of spinach chloroplasts. Biochemistry 11: 1155-1160. 4. Gotta. J. .\I.. R. CArHsa. Asp G. D. \VINGET. 1972. Advantages of the use of (.‘a-rnkuv counting fur the .lvtermtnatmn of -'”-'P in I‘llult)[llllnpllnrylnllon ra- aenrch. Anal. Biochem. 50: $40-$48. . Goran. J. M. Axn S. szvVA. 1913. Photosystem II electron transport and phos- phorylation with dibromothymoquinone as the electron acceptor. Eur. .1. Biochim. 37: 185-192. 0. Gone. .1. M. tsp S. IzAwA. 1973. Studies on the energy coupling sites of photo- phosphorylation I. Separation of site I anal II by partial reactions of the chloroplast electron transport chain. Biochim. Biophys. Acta 314: 211-223. 7. GOULD. J. .\'I. .\.\'D D. R. 031'. 1973. Studies on the energy coupling sites of photophosphorylation III. The different effects nl methylamine and ADP plus plttmphate on electron transport through coupling sites I and II in 130- lntnl chloroplasts. Blot-him. Biophys. Acta 325: 157-166. 8. HINT). G. AND A. JAcaxoonr. 1965. Separation of light and dark stages in pho- tophosphorylation. Proc. Nat. Acad. Set. I'..\'..-\. l9: 715-722. . ItAwA. S. 1968. Effect of Hill reaction inhibitors on photosystem I. In: K. Shi- bata. «1.. Comparative Bttwhenustry and Biophysics or Plin-tOsyntthIs. Uni- versity Park Press. State College. Pa. pp. 140—147. 10. ll.\WA. 5.. T. N. CONNOLLY. G. D. thca‘r. Axn N. E. Goon. 1909. Inhibition and uncoupling of photophosphorylation In chloroplasts. Bro-)khnven Symp. Biol. 19: 169-187. 11. IzAwA. S. no N. E. Goon. 1969. Effect of p-chloromercuribensoate sud mercuric ion on chloroplast photophosphorylation. Progress in Photosyn- thesis Resesrch. Vol. III. pp. 1288-1298. 12. Ian". 8.. J. M. Gocu). D. R. Orr. P. Flues. Axe N. E. Goon. ms. Elec- tron transport and phosphorylation in chloroplasts as a function of the elec- tron acceptor III. A dibromothy- - _--_.:tvc phosphorylation asso- cratsd with photosystem II. Biochim. Biophys. Acta 305: 110-128. 13. limitless. M. xxn S. KATOH. 1912. Studies on electron transport associated with photosystem II. Functional site of plastocyanin: inhibitory effect of HgCl: on electron transport and plastocyanin in chloroplasts. Biochim. Biophys. Acta 283: 208-278. M. KaAArasuor. R., S. lewA. asp B. CHANCE. 1912. Use of uncoupling acridine dyes as storehromctric probes in chloroplasts. Plant Physiol. 50: 713-718. 15. Laura. A. W. D. Axe W. D. Bosses. 1912. The effect of artificial electron donor and acceptor systems in light-induced absorbance responses of cyto- chrome I and other pigments in intact chloroplasts. Biochim. Biophys. Acta 287: 149— 159. 10. Muss. D.. P. Roux. S. FAaAc. R. Goomx. J. Lt'rz. A. Mount-A. 8. Ron- stct‘u. Asp C. “text. 1913. Hrt-A DCMU independent electron acceptor of photosystem II. Biochem. Biophys. Ru. Commun. 50: 1113—1119. 17. Oar. D. R.. S. lsAwA. N. E. Goon. AND D. W. Kaocsuxx. 1973. The efl'ects of the plastocyanin antaaonists KCN and polyoL-lystnc on partial reactions in isolated chloroplasts. FEBS Lett. 31: 110-122. 18. 01'1TIAKL'L. R. AND 8. IwaA. 1913. Electron transport and phosphorylation in chloroplasts as a function of the electron acceptor II. Acceptor-specific in- hibition by KCN. Biochim. Biophys. Acta 303: 1015-118. 19. Sun. 8.. R. Ocrmxct. S. IuwA AND N. E. Goon. 1971. Ell't‘ll'fln transport and phosphorylation in chloroplasts as a function of the electron acceptor. J. Biol. Chem. 240: 3206-8209. Tsassr. A. Ass 8. Return. 1913. Properties of photoreduction by photosystem II in isolated chloroplasts: an energy-conserving step in the photoreduction of benzoquinone by photosystem II in the presence or «libromothymoquturme. Biochim. Biophys. Acta. 3&5: 120-139. 21. Wilmer. G. D.. S. IaAwA. Asa N. E. Goon. 1960. The inhibition of photophos- phorylation by phlorizin and closely related compounds. Biochennstry 8: 2007-2014. 01 0‘ 2 . O APPENDIX VI STUDIES ON THE ENERGY COUPLING SITES OF PHOTOPHOSPHORYLATION IV. THE RELATION OF PROTON FLUXES TO THE ELECTRON TRANSPORT AND ATP FORMATION ASSOCIATED WITH PHOTOSYSTEM II Reprinted from Biochimi'ca er Bi0p/iysi‘ca Acta. 333 (1974) $09—$24 LC) Elsevier Scientific Publishing Company. Amsterdam - Printed in The Netherlands 83A 46717 STUDIES ON THE ENERGY COUPLING SITES OF PHOTOPHOSPHOR- YLATION IV. THE RELATION OF PROTON FLUXES TO THE ELECTRON TRANS- PORT AND ATP FORMATION ASSOCIATED WITH PHOTOSYSTEM II J. MICHAEL GOULD and S. IZAWA Department of Botany and Plant Pathology, Michigan 5mm Uni'i'crsiry, East Lansing, Mir/i. 48824 (U.S./1.) (Received September 24th, 1973) SUMMARY 1. By using dibromothymoquinone as the electron acceptor. it is possible to isolate functionally that segment 01' the chloroplast clcctron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (COUpling Site 11. obscrvcd Pic: . . (1.3-0.4). A light- dcpcndcnt. reversible proton translocation reaction is associated with the clcctron transport pathway: H20 —+ Photosystem ll .1 tlibromothymoquinonc. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site 11. 2. The initial phase of H+ uptake. :iiialy/cd by :1 flash-yield technique. cx- hibits lincztr kinetics (0-3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush") cncountcrcd in thc ovcrull Hill rcaction with methyl- viologen. Thus the initial rate of H‘” uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH chzingc tracing obtained under continuous illumination. 3. Dibromothymoquinonc reduction, obscrxcd as O: cvolution by a similar flash-yield technique. is also linear for at least the lirst 5 s, the rate 01' Oz cvolution agreeing wcll with the stczidy-statc l’ilIC obscrvcd undcr continuous illumination. 4. Such measurements of the initial rates 01‘ O2 cvolution and i-i+ uptake yield an H+/'e’ ratio close to 0.5 for thc Photosystcm ll partial reaction regardless of pH from 6 to 8. (Parallel experiments for thc methylviologen Hill reaction yield an Hfl'c” ratio ol‘ 1.7 tit pH 7.6.) 5. When dibrommhymoquinonc is being reduced. concurrent phosphoryl- ation (or arsenylation) markedly lowers the extent of H+ uptake (by40-60 Sf, ). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to Abbreviations: P/c3. ratio of the numbcr ot' molcculcs of ATP l'ormcd to the number of pairs ofelcctrons transported; H *,’c‘. ratio of thc numbcr of hydrogen ions transporicd to the number of electrons transported; DBMIB. 2,5-c’ibromo-3-mcih_vl-o-isopropyl-p-bcnzoquinonc (dibromothymo- quinone). 122 510 an unequivocal interpretation since phosphorylation does not alter the rate of elec- tron transport in the Photosystem ll partial reaction. ADP. Pi and hexokinase, when added individually, have no effect on proton uptake in this system. 6. The involvement of a proton uptake reaction with an HU’e' ratio of 0.5 in the Photosystem II partial reaction H20 -» Photosystem ll —. dibromothymoo quinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/ez) and proton uptake (H+/e‘) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of e ergy coupling. u--.— m0w-..—..——.--.-a _ -M . ... - ._. M INTRODUCTION The electron transport pathway in isolated chloroplasts can be divided into several segments by the use of appropriate electron donor—acceptor combinations in conjunction with specific inhibitors of certain intermediate electron carriers [l]. When this is done it becomes clear that there are two sites of energy conservation in the phosphorylation-coupled transport of electrons from water to Phomsystem l [l—S]. These two coupling sites dill'er markedly in their characteristics. Coupling Site 1 refers to the energy conservation site associated with the transfer of electrons from plastoquinone to cytochromef [6, 7]. This site provides the rate-determining step for the overall Hill reaction [l l. The rate of electron flux thrOugh Coupling Site I is greatly increased by ADP plus phosphate under phosphoryl- ating conditions and still more by the presence of phosphorylation uncouplers. The efficiency of phosphorylation (Pfez) at Site l is dependent on the pH of the medium. with an optimum at pH 8-8.S where the observed P/e2 is 0.6-(ft? (ref. 3). ATP for- mation supported by Coupling Site I is inhibited by the chloroplast energy transfer inhibitor HgClz (ref. 8). . Coupling Site II is associated with the transfer of electrons from water to plastoquinone or with the transfer of electrons from water to exogenous lipophilic “Class III" oxidants which intercept electrons before they reach plastoquinone [5, 9]. Coupling Site ll dilfers from Site I in a number of important respects. The rate of electron transport through Site [I is independent of phosphorylation and uncoupling [l, 9, 10]. The elficicncy of phosphorylation is practically independent of pH (6—9). and the observed P/ez ratio is characteristically 0.3—0.4 [1—3. 5. 9. 10]. Moreover. ATP formation supported by Coupling Site II is insensitive to HgCl2 (ref. 8). These differences suggest that the modes of energy transduction at Sites I and II are somehow different. Therefore we have been prompted to undertake a further investigation into the mechanism by which electron transport may be coupled to phosphorylation at these two sites. This paper deals primarily with further charac- terizations of the light-induced proton uptake (pH rise) associated withthe transfer of electrons from water to dibromothymoquinone via Photosystem l1 [1]. ln pre- vious papers [3, 9, 13] we established that this plastoquinone antagonist [l l ], which at low concentrations (0.5 pM) blocks the transport of electrons from reduced plastoquinone to cytochrome] [12}. can act at higher concentrations (25 'lM) as an acceptor of electrons from Photosystem ll, probably via plastoquinone [9]. In l23 5” addition, we have shown that the phosphorylation reaction associated with dibromo- thymoquinone reduction exhibits all of the characteristics of Coupling Site ll out- lined above [9]. The use of substrate concentrations of dibromothymoquinone provides a con- venient reaction system for studying the nature of Coupling Site ll, since the function of the substrate as an inhibitor effectively blocks further electron transport (to Photosystem l) and thereby isolates Site II from Site I. Furthermore, we have recently presented evidence that a reversible “proton pump" is associated with dibromothy- moquinone reduction [l]. Since electron transport in this system is unaffected by uncouplers or by phosphorylation, it has been possible to observe directly the effects of ATP formation on the “proton pump" without a complication encountered by previous investigators [14, 15]. Herctofore it has been impossible to study the effect of phosphorylation on proton uptake without increasing the electron transport rate. thereby inadvertently modifying the associated proton uptake. Therefore it has previously not been possible to study the proton pump and phosphorylation as opposing processes in an unambiguous way. The results presented in this paper show that there is a quantitative rela- tionship between the electron transport-dependent accumulation of hydrogen ions and ATP formation at Coupling Site ll. The results also suggest that Coupling Site II in chloroplasts may be identified with the water-oxidation reaction, which seems to release protons to the inside of the chloroplast lamellar membrane. MATERIALS AND METHODS Chloroplasts (intact, naked lamellae) were isolated in the cold from leaves of fresh market spinach (Spinaeia oleracea L.) as described in a previous paper in this series [I]. For some experiments the HEPES—NaOH buffer in the suspension medium was replaced with either phosphate or arsenate and the bovine serum albumin was omitted. The concentration of chloroplasts in the final stock suspension was adjusted so that the amount of buffer carried over into the reaction mixture gave a final concentration of 0.5 or ID mM. Changes in hydrogen ion concentration in the reaction mixture were detected with a Corning semi-micro (“Tri-purpose") combination pH electrode connected to a fast responding Heath/Schlumbcrger EL’ZOO-Til.) pH electrometer module equipped with a Heath/Schlumbcrger EUZOO-OZ DC offset module to facilitate scale expansion. The output from the electrometer was recorded on an Esterline-Angus strip chart recorder. The response half-time for the pH measuring system was in the order of 0.5—l s. Changes in pH were normally monitored with a scale expansion of 0.l pH unit full scale (l0 inches) on the recorder. The noise level at this amplification was less than 0.002 pH unit. In routine pH experiments. reactions were run in a final volume of 2.0 ml in thermostated vessels at l8 "C with continuous stirring. Prior to illumination the reaction mixture was adjusted to the desired initial pH with small volumes of dilute NaOH or HCl. Actinic illumination was supplied by a SOD-W slide projector. The beam was passed through a SOO-ml round-bottomed flask containing a dilute CuSO4 solution (which served both as an infrared tilter and as acondensing lens)and through an orange glass filter (transmission ;~- (ill-i) nm) plus a Corning l-(i‘) heat filter. The 124 $2 light intensity was approximately 700 kergs -s‘l - em'2 (600—700 nm). At the end of each experiment the pH changes registered on the chart paper were translated into H” equivalents by titrating the reaction mixture in the light with a known amount of 0.001 M HCl. Electron transport was measured as oxygen evolution (dibromothymoquinone as acceptor) or oxygen uptake (methylviologen as electron acceptor) [16] using a Clark-type membranocovered oxygen electrode. For these experiments a 3.0-ml reac- tion mixture was used. When both electron transport and proton uptake were deter- mined, the pH rise was measured in an identical reaction mixture in the same appa- ratus, substituting the pH electrode for the oxygen electrode. RESULTS Light-induced pH rise associated with dibromot/tynmqm’none reduction We have previously presented evidence that the electron transport pathway H20 --> Photosystem lI -9 dibromothymoquinone, which includes Coupling Site II but not Coupling Site I, is associated with a light-dependent, reversible proton up- take [l]. These results have been confirmed and extended (Fig. I). Fig. 1 depicts the general pattern of the light-induced pH rise in the medium. Above pH 8.l, where reduced dibromothymoquinone is rapidly reoxidized by mole- cular oxygen [9] (and therefore no exhaustion of the electron acceptor occurs). the pH rise can be observed many times using repeated light cycles (trace A). Below pH 8, where the reoxidation rate is very slow. the pH shift can be maintained only as long as the reduction of dibromothymoquinone continues. As the reduction approaches completion and the electron transport slows down. a gradual reversal of the pH change is observed even in the light. and eventually the pH. returns to the original level (trace 8). Subsequent illuminations do not restore the pH rise. If the light is turned off before the complete exhaustion of the electron acceptor. a second light cycle does induce a small pH rise. the extent of which depends upon the amount of oxidized dibromothymoquinone remaining. The uncoupler gramicidin D (Ling/ml) completely abolishes the light-induced pH response (data not shown _). lf ferricyanide is added to the reaction mixture so that the photoreduced dibromothymoquinone is continually reoxidized by the excess ferricyanide. a revers- ible pH rise superimposed on an irreversible pH drop is observed (Fig. l trace C). This is repeatable even below pH 8 since the reoxidation of reduced dibromothymo- quinone by ferricyanide is very rapid at any pH above 6. The irreversible pH drop is due to the protons produced by the oxidation of water according to the equation: l/2 H20+Fe“ —» 1/4 02+Fe“4—H*. Thus the rate of proton production can also be used as a means for determining the rate of electron transport. As trace D shows, both the transient pH rise and the lag in the light-induced pH drop due to the superimposed proton uptake are eliminated by the uncoupler gramicidin, although the rate of electron transport (measured as proton production) is scarcely affected. This confirms our previous observations that the rate of electron transport associated with dibromothymoquinone reduction is not increased by uncouplers [9]. l25 513 F—-——-f T A) I I0 sec ' IO nelguiv. 0.02 pH 1. l 3 initial pH- i 8.l5 1 l 1 light on i lightoff l t 8) increasing pH 7.30 4 . 1 y T T l0 ne‘cwiv. l .02 pH .1. l _/ i 7.25 1 1 t———ot l5 sec C) COMtOl D) o gramicidin 0 initial pH = 725 7.25 T 50 netwiv. i decreasing pH 20 see Fig. l. Light-induced pH changes associated with the partial electron transport pathway HzO —- Photosystcm Il -’ dibromothymoquinonc in isolated chloroplasts. 'l‘he 2.0-ml reaction mixture con- tained 0.! M sucrose. 2 mM MgCl1, 50 mM NaCl, 0.5 m M lllil’liS-VafHI buffer, loiiM dibromo- thymoquinone and chloroplasts containing IOO ‘ug chlorophyll. in the experiments presented in traces C and D. 0.5 mM potassium ferricyanide was added to the reaction to continually reoxidize the photo- reduccd dibromothymoquinone. Note that the rate ot'elcctron transport (PM; in VHCQUIV - h“ - mg chlorophyll") is not increased by the uncoupler gramicidin D. For further explanation see the text. Stoichiometry between electron transport and proton uptake (H ‘;'e') The data presented in Fig. l are most easily interpreted in terms of a trans- membrane H+ gradient associated with the Photosystem ll partial reaction H20 _. Photosystem ll _. dibromothymoquinone. However. the significance of this proton gradient and its relation to the coupling mechanism cannot be evaluated critically l26 5l4 until the efficiency of proton uptake (H+,"e") is determined. Moreover, determination of H‘Vc‘ in this case is of special interest, since the dibromothymoquinone partial reaction includes only one (Site ll) of the two coupling sites associated with the complete Hill reaction [3, 9]. Thus the observed efficiency of ATP formation (P/e2) supported by this partial electron transport pathway is lower than the efficiency of the complete chain where both coupling sites are operating (P/e2 :- 0.3-0.4 versus l.l-l.2, respectively) [1]. Therefore one might reasonably expect the efficiency of proton uptake associated with Site II to becorrespondingly low. However, the deter- mination of an HU’e‘ ratio involves a variety of difficulties, and. in fact. no truly unequivocal method of measuring it has as yet been developed. For a more complete discussion of these difficulties, see review articles by Jagendorf [17], Walker and Crofts [l8] and Schwartz [l ]. In the experiments outlined below we have observed the initial kinetics of proton uptake and electron transport by two different methods in an attempt to circumvent these difficulties and to accurately measure the H+;’e” in the dibromothymoquinone partial reaction. The response time (t, S l s) of the pH assay system used in these experiments (see Methods) was considerably faster than the apparent kinetics of the pH rise (’t 2 4—5 s) and therefore it seemed possible that the relatively linear initial phase I .T i" . i i l vi I ~:--~ :~- -i~~—~- ~ -—.~——a:-—————-~f— ? . t l ’ . I ' ' t i _i i t I LT f ' t 7 —' U, ' . i L ' .1 l- ___ __ " ‘f‘ ‘ i“”*"' I ‘ 7"“i 'i '. ' ' I I .' ‘ l * T I , : : g . l5 sec '3; - 0.0. pH . - i -~ -. -~ r'" ~- - -'-- a - -~--*--* -—~-—--;"~-oii + T T ‘6, I I , I I '5 - Q i »--. - v-I- -— -»» —————. . _,_._.__.__.-_-_.____-.. b—-—o . - - l l ' i . l5 neouw. H' ‘,' O” __.-.__ .——‘.————7_ ' l I t: l .. _. __, .......4..-_ .-. . -.. C‘- .‘_ - ..;..__- .,. .-.-... AHO‘IZS l J - l g ', ....... .i-.u—— -4“.-- . I i i L . 1‘ _L__.._._. _.—. ___ “w 5 mm...“ ' l l .-...._..|__.__.--‘ . - _._ _--._.._____ _-1__.,___T _im- _ ..- Fig. 2. Recorder tracing of the apparent kinetics of the reversible pl-l rise associated with Photo- system If electron transport from water to dibromothymoquinone. The reaction mixture was as in liig. l, trace A. except the lllil’LS buffer was replaced by 2.25 m M Na;lll’l )4 and contained chloro- plasts equivalent to I45 [lg chlorophyll. The :nitial pll was 8.”). Numbers in parentheses are the apparent initial rates ofproton uptake in peuuiv f-l * ~ It " - mg chlorophyll ‘ '. The steady-state C\tcttt ofthe pH rise (. lll '; in ncquiv - mg chlorophyll") “as determined by titrating the reaction mixture in the light with known volumes of 0.00l ,Vl llCl (not shown). ".27 515 of' the pH tracings (Fig. 2) might provide a reasonably accurate estimate of the ini- tial rate of‘ proton uptake. This assumption would be invalid, of‘ course, if the pH rise involved any transient kinetics faster than the instrument response time, such as the initial “pH gush" (’t 2: 0.10 5) described earlier by Izawa and Hind [20]. In order to detect the pOSSibiC involvement of such transient kinetics, we have examined pH changes induced by a series of brief‘ illuminations (flash duration l-3 s) with inter- vening dark periods to allow for the lag due to instrument response time. That is, a sufficient dark period was introduced between successive flashes to allow the insuu- ment to record the entire pH change which had been completed during the flash. This dark period was not long enough to allow any significant decay of the pH change, however. Somewhat surprisingly, we could detect no evidence for any sig- nificant “gush“ phenomena at all associated with dibromothymoquinone reduction (Fig. 3, trace b). the rate of' proton uptake determined for the first flash being essen- tially the same as :he rate determined for a subsequent flash. Plot c of Fig. 3 represents the early phase of‘ pr0ton uptake reconstructed by summing the flash yields and illu- mination times of trace b. A very good agreement was found between the initial slope of'the linear phase ol'the reconstructed time-course curve (< 45; trace c) and the initial slope of' the continuous recorder tracing (trace a). This is not surprising since, as trace b shows, there was no significant overshoot of' the pH response after a flash except for the small amount attributable to the instrumental time lag. Nor was there a rapid enough dark decay to obscure the flash yield. In other words, each flash yield determination did seem to accurately represent the extent of the pH rise which had been essentially completed during the flash. Thus the initial slope ofa pH rise curve obtained under continuous light must in fact be a fairly reliable measure of‘ the actual initial rate of. proton influx. The rate of‘ electron transport in the same time range (O«5 s after light on) was measured as O; evolation using a duplicate reaction mixture in the same apparatus (Fig. 3. traces d 1'). Flash experiments were even more important in this case, be- cause of‘ the very slow response time (t. 2; 2 s) of' the membrane-covered oxygen electrode used. The flash experiments again detected no sign of‘ transient phenomena (Fig. 3, trace e) and again the reconstructed slope agreed rather precisely with the steady-state slope determined under continuous light (traces d, l'). (Transient kinetics such as those discovered by Joliot et al. [2! l are far beyond the resolution of our in- strument.) The ratio H T "e' determined from the data presented in this set of experi- ff‘tc‘flfs‘ (Hg. 3) outs U.Sf. The use of intermittent brief periods of' illumination described above makes transient differences between initiai rates and steady state rates quite obvious when these differences do exist. ThUs the initi; . “pH gush" known to be associated with the reduction of methylviologen 'o) chloroplasts {20! is clearly revealed by the flash tech- nique (Fig. 4b) even though the pH changes recorded during continuous illumination conceal the transient (Fig. 4.1). However. if' the tracing for the continuous illumina- tion is corrected for the instrument response time according to the method of“ Izawa and Hind [20}. there is good agreement between the kinetics of the pH rise as deter- mined by the flash ‘ytcld technique and the corrected kinetics for continuous illumi- nation. The "pll gush” amounted to one mole of H * for each 2t) 30 moles chlorophyll in this experiment. However. no corresponding initial last phase in oxygen production could oe detected (Fig. 4b), indicating that the “pH-gash" observed under some l28 516 (cl X, i x / l 0.2T , o ! / . i I 6px, i 3 e,’ l g ~ ,’° 3 z I,/ l u I s / l EOII- / l .25 0 l 3 . O" Q) a _ l i 0 o/J ' L f L i O 4 8 t seconds ) i / O i' 0.6 if (f) E i- 0 Q. 8 0.4- 2 \ i f. 3? U. I. \ E o \ 3 i g 0.2)- l ‘3’ i =- l _ o l o L t g t I L . O 4 8 :2 | IO sec seconds l 4 Fig. 3. lnitial kinetics ofthe light induced pH rise and electron transport measured under both flash and continuous illumination with dibroniothymoquinone as the electron acceptor. The reaction n'f!.\- ture (3.0 ml) was as in Fig. l, trace :1, except that the concentration of' dibromothynioouinoiie was 50 [IM and the reaction contained chloroplasts equivalent to ISO/lg chlorophyll. The initial pH was 7.4. Trace a: recorder tracing ofthe apparent initial kinetics of‘ the pH rise observed under continuous illumination. The rate of' H ’ uptake in [ICQUlV H ‘ - h " - mg chlorophyll “ was determined from the essentially linear initial portion of the tracing and is shown in parentheses. Trace b: recorder tracing of the pH rise induced by flash illumination. Numbers in parentheses represent the flash duration (in 5). Trace e: reconstructed time course of‘ the initial portion of‘ the pH rise from flash yield determi- nations (trace b). Note that the rate of proton uptake measured by the flash technique agrees very well with the rate determined under continuous illumination ttrace a). Trace d: recorder tracing of the kinetics of‘O; evolution under continuous illumination. The rate of‘electron transport (in ,iiequiv - h "’ - mg chlorophyll") is shown in parentheses. Trace e: recorder tracing of' 0; evolution measured by the flash yield technique. Numbers in parentheses represent flash duration (in seconds). Trace t‘: reconstructed time-course for 0, evolution determined from the flash experiment (trace e). Note that the rate determined by this technique agrees sery well with the steady-state rate determined under continuous illumination (trace d). l29 Us ‘1 F l o) HZO-d-MV i bl H20—-MV c) H204DBMIB ' / i 30 ' l- i- e r .‘ '1 0 i- / ' (stecdy- '7 l 3 AH . /° stoic) '- | .— 0 ' E "III”, OH. 5 \ on’ /o/ 'i’ q, (mm ("00?!) I I .' ‘. C 20 " O r" \ // ’1‘, i‘ E T .I ‘ 0’ 9 a? .u' . (hull) ,' / .— . ' ‘ ‘ 8 / x a v. \: a» (comm- , . tuner . \ P < O alum.) I 0' ’I .'.'.' ‘ .‘ 'l d . Io - l.- l I k ‘ ‘ .2 / . / , t, . . 3 i I .. ' 13H U i " " )\ E Y 'I (continuous g I 4/("o’h’ IIIUM ) on [on 3’ in i ’ ‘ ‘ O \ 1 1 1 1 i 1 1 1 1 \L’1 1 1 1 i 1 1 1 1 l \ 1 1 L #1 1 1 O 5 l0 0 5 l0 0 5 lo TIME (seconds) Fig. 4. Comparison between the initial rates ol‘proton uptake and electron transport for the complete electron transport chain (H20 —> methylviologen) and the Photosystem ll partial reaction H30 ~- dibromothymoquinone. Reaction conditions were as in Fig. 3 with the following exceptions. In the experiments presented above in a and b, dibromothymoquinone (DBVHB) was omitted and I00 p31 methylviologen (MV) was added. When dibromothymoquinone was the electron acceptor. electron transport (E.T.) was observed as 0, evolution. When methylviologen was the acceptor. electron transport was followed as O, uptake [l6]. The chlorophyll concentration was Song/ml. (a) Compari- son between the observed pH change (:lH +) under continuous illumination and by a flash yield tech- nique. Note that the large "pH gush“. which is clearly C\ItiL‘lll in the kinetics obtained by time flash technique. is masked in the continuous tracing due to the instrumental response lag. Nevertheless the slope of the relatively linear portion of the pll rise immediately following the "gush" is in good agreement with the slope obtained with continuous illumination. The initial pH For this experiment was 6.8. (b) Comparison between the kinetics of H' uptake (. lH'l and electron transport (E.T.) when methylviologen is the electron acceptor. The data shown is l'rom two identical experiments done at pH 7.6. Note that the rate ol'electron transport determined under continuous illumination (dashed line) agrees very well with the electron transport rate determined by the flash technique (points). and that there is apparently no initial burst ol‘eleetron transport corresponding to the initial "pH gush“. The H’l'e" ratio determined for this experiment (see text) was l.7. tc) An experiment similar to b above except the electron transport pathway included only Photosystem ll and only Coupling Site ll. Dibromothymoquinone served as the electron acceptor. Note that the initial "pH gush" is absent in this system. The H’le' ratio calculated for this experiment ‘pH ".2) “as 0.45 (See also Fig. 3). conditions cannot be simply explained in terms of an initial rapid rate of electron transport associated with plastoquinone reduction. The ratio H Kc ’ observed during methylviologen reduction (calculated from the relatively linear portion of the early phase of the pH rise after the initial gush) was L7. .\'ote again (Fig. 4c) that there was no signilicant initial rapid phase of proton uptake when dibromothymoquinone was being reduced and that the ratio H+’e' was much lower. about 0.5. Thus the proton pump associated with dibromothymoquinone reduction (involving only Coupling Site ll) is distinguished From the proton pump associated with methyl- viologen reduction (involving both Sites l and H) in two ways: the reaction involving only Site ll lacks an intitial rapid phase and is less than ball as ellicient as the com- bined sites in transporting H+ across the lamellar membranes. Table 1 summarizes the results ol‘ three independent series of experiments performed with diflerent chloroplast preparations. While the ratio HU’e’ for the dibromothymoquinone reducing system is relatively constant (0 35—O.5) over a wide l30 518 TABLE I STOICHIOMETRY BETWEEN PROTON UPTAKE AND ELECTRON TRANSPO RT (H‘lc‘) WITH DlBROMOTHYMOQUlNONE OR METHYLVIOLOGEN AS ELECTRON ACCEPTOR Reactions were run as described in Figs 3 and 4. The basic reaction mixture was as in Fig. 5 exec-pt in Expt 1, where the phosphate butl'er was replaced by 0.5 mM HEPES-NaOH. The chlorophyll concentration was IOO [igglmL Note that when dibromothymoqumone served as the electron acceptor so that only Coupling Site ll was being utilized, the ratio H‘le‘ was relatively constant (0.35—0.5l) over a wide pH range. When methylviologen served as the electron acceptor (utilizing Coupling Sites land ll). the H*,'e‘ ratio was much higher. - —..- ..»_..-_-.—.._._.—.__. .. . . -..-——___.__—. . - -_. __,. - .. _“v- ._..._. __ __ .--__._ -.. Expt Electron lnitial pH lnitial rate (‘IICQUiV - h'l - mg chlorophyll") H‘ie‘ No. acceptor -- -'--~- - - -- - » --~ - '- H * uptake Electron transport l Dibromothymo- 6.22 93’" 2| 8** 0.42 quinone 6.60 l02 225 0.45 7.00 R9 234 0.38 7.43 3| 2l2 0.38 2* Dibromothymo- 7.20 100'M 198“ 0.5l quinone 7.60 63 146 0.43 8.00 48 I25 0.38 3* Dibromothymo- 7.30 69” l72** 0.40 quinone 8.l3 60Mr loft“ 0.36 8.l5 S3 lS3 0.35 Methylviologen 7.40 103‘" 59'” l.74 7.75 [OSMr 63’” .7l vhmwMG-t -n— . . ‘o—c-uu- M- . * Phosphate bull'er (2.5 mM). ** Measured by flash yield determinations. Other values are From continuous tracings. range of pH values (62-83.), there is some tendency For the lower H+/e‘ values to be associated with the higher pH values. Several considerations lead us to believe that the true elliciency of proton translocation in this system is even more pH-independent than actually observed, however, and that the true value for the ratio H+/C_ is most likely about 0.5, the highest value actually encountered at the lower pH regimes (pH 6-7). At higher pH‘s, especially above pH 8, the outward dichsion of protons in the light is probably much faster and therefore competes more efliciently with proton accumulation (uptake). This was apparent in the experiments. since the dark decay process observed after turning oil the light was approximately twice as last at pH 8 as at pH 7 (decay half-time r: 7 sat pH 8; l5 sat pH 7). It was also appar- ent in the flash experiments that the initial linear phase of the pH rise at pH 3 was somewhat shorter than at pH 7.3 (see Fig. 3. trace c). Eflec! of phosphorylation and arsenylation on pro/mi uptake Both the chemiosmotic and the chemical coupling hypotheses predict that concomitant phosphorylation or arsenylation should decrease the steady-state ex- tent ol' the proton uptake [22]. Several workers have indeed observed a decrease in the pH rise by concomitant phosphorylation or arsenylation [l5,-3}. but the opposite elTect -— a stimulation of proton uptake by arsenylation —- has also been reported [14]. As has been pointed out by Dilley and Shavit [IS], the acceleration of electron transport in ordinary noncyclic electron transport systems by phospho- l3l 5l9 rylation (or in some cases by the elTect of ions in the phosphorylation medium, e.g. Mg“), and resultant increases in the rate of proton flux could easily mask the true effect of phosphorylation on proton gradients. Moreover, the addition of ADP (or ATP) itself has also been shown to cause marked increases in proton uptake [23]. However, by using the Photosystem ll-dependent dibromothymoquinone reduction one avoids these complications since the electron transport in this system is not stim- ulated by phosphorylation [9], nor is proton uptake enhanced by the addition of ADP (cf. Table ll). This reaction therefore provides a convenient system in which to test the elTect of phosphorylation on proton uptake. TABLE ll EFFECT OF PHOSPHORYLATION AND ARSENYLA’l‘lt)N ON THE EXTENT OF THE LIGHT-INDUCED PROTON UPTAKE ASSOClATH) \\'lTll 'l'llLL ELECTRON TRANSPORT PATHWAY H10 ._, PllOTOSYSTEM ll -1 DIBROMOTll\".‘vl()QLilN()NE Reactions were run as in Figs 5 and 6. Final concentrations of the additions were: Pr, 2.5 mM; ADP. 7534M; hexokinase, lygilml 1plus 30 mM glucose): HAsOf‘. 2.5 m M. The extent of the proton uptake \s as determined by titration in the light as described in Methods. Note that the presence of a complete phosphorylation or arsenylation system significantly lowers the steady-state extent of the proton uptake, although the individual phosphorylating agents haxc no ell'cct by themselves (Exp! l). Expt lnitial pH Additions .lll ' extent (“.L) No. neouiv,‘mg chlorophyll l 8.14 None 42 (l00) A Dl’ 42 (low Hesokinase 40 (99) ADP, Pi, hexokinase 26 (62) 2 8.l5 Pt 56 (l00) ADP, Pi, hexokinase 22 (~10) 3 8.22 Pl . 70 (100) ADP, Pt, hexokinase 39 (56) 4 8.27 HAsOJ ' 55 (100) HA5042-, ADP 4': (SO) 5 8.25 HAsO.,2 ‘ 73 HOW HAso.’-, ADP 59 (Si) —— .... ._ in Fig. 5, trace A demonstrates the reproducibility of the pH rise in a phos- phate-containing suspension over repeated light cycles. This reproducibility greatly facilitated the experiments since the elTect ol’ an adt’itiye (e.g. ADP)could be exam- ined without preparing a new reaction mixture. Trace B shows that in the absence of hexokinase, the addition of ADP initiates ATP formation which can be followed as the irreversible consumption of protons according to the equation: ADP3‘+HPO,2'+H* _. ATP4‘4-HZO (pH 3) Clearly the extent of the reversible proton uptake (seen as a proton elllux after turning 00' the light) is smaller under phosphorylating conditions (' -- ADP) than under non-phosphorylating conditions ( ADP). This is demonstrated in a dill'erent way in trace C, where the hexokinaser- glucose system is used to consume the ATP 520 i ) l T M increasing pH A) .I 8 5 { ‘ AH‘: IO3.5 97.3 9I.o T L/_ _____________ 0.0l pH 1 l 0.. ‘ ADP A“ "E‘ftfl ‘L B) k l 8.l4 . 1 light on l i———-i I5 sec. 1- ‘ ADP, : hexoxinose IO neouiv. .l 8 3 ‘ 4 1 AH*- "7.0 60.3 54.7 Fig. 5. ElTect of phosphorylation on the extent of the light-induced proton uptake associated with electron transport from water to dibromothymoquinone. Reaction conditions are as in Fig. l, trace A, except that the HEPES bufl'er was replaced by 2.5 mM NazH P04. The extent of the proton uptake (Al-1") was determined as described in Methods. Trace A illustrates the repeatability of the pH rise over repeated light—dark cycles. Trace B shows that the addition ofADP (linal concentration 75 [ltVU causes an irreversible proton consumption in the light (due to ATP formation) and results in a decrease in the extent of the reversible proton uptake (seen as a dark elllux after turning otl‘ the light). This irreversible proton consumption was eliminated in trace C by the addition of hexokinase (_l ,ig/Zml) and glucose (IO mM) with the ADP (75 ,uM). Again a significant decrease in the extent of the proton uptake is observed. (The additives themselves had only a negligible etfect on the bull‘ering capacity of the reaction mixture.) as it is produced and thus eliminate the irreversible proton consumption due to ATP formation. Hexokinase alone has no clfect on the extent of the pH rise (cf. Table ll). If arsenate replaces phosphate (with or without hexokinasc) a situation simi- lar to the ADP-i-Pi-l—hexokinase system should be observed. since the unstable ar- senylated ADP does not accumulate. Fig. 6 shows that indeed this is the case. A marked lowering in the extent of proton uptake is observed upon addition of ADP to an arsenate containing medium (in this case without hexokinase). Table ll summarizes the elTect of various additions on the extent of the proton uptake associated with dibromothymoquinone reduction. it can be seen that in this system ADP, hexokinase or phosphate alone have no elTect on the extent of proton uptake. The lack of effect by ADP in this system is in contrast with the observation of McCarty et al. [23]. Using the methylviologen system, they too n0ted that hexo- kinase had no effect on proton uptake, but they did observe a large stimulation by low concentrations of ADP. We have confirmed their observation using methylvio- logen as electron acceptor, although in our chloroplast preparations the maximum stimulation obtained was only 3040““. The complete lack of ADP elTect on proton uptake in the partial reaction involving only Coupling Site ll suggests that the stimu- 521 l l l . A 8.25 _J to nequiv. ) 1 1 1 AH’ = 72.5 68 2 0.02 pH i 1 ATP { ‘, 8 I5 _/\‘ M AH" = 70.3 43.7 474 increasing pH light on 1 light oitl '5 "a" Fig. 6. LtTect ol'arscnylation on the extent ol‘the light induced proton uptake associated with electron transport to dibromothymoquinone. Reaction conditions were the same as in Fig. 5. except the Na,- HPO.t butler was replaced by 2.5 mM NalHAsO‘ and no ltexokinase plus glucose was added. Netc that the extent of the H * uptake was signiticantly smaller when ADP was added to the reaction mix- ture. The arsenylation ofADP does not result in an irreversible proton consumption, however, since the arsenylated nucleotide hydrolyzes rapidly. latory ell'eet of ADP may be expressed only when the electron transport system in- cludes Coupling Site 1. DISCUSSION The characteristics of the proton pump associated with the electron pathway H20 —~ Photosystem ll —. dibromothymoquinone, revealed in this study, may be summarized as follows: (i) approximately one proton is taken up for every pair of electrons transported (or H+,’e‘ ratio 0.5; observed range, 0.4-0.5), (ii) the elf]- ciency of proton uptake is essentially pH-indepcndcnt (between 6.2 and 8.2), and (iii) the kinetics of proton uptake, as observed by flash-yield determinations, Show no sign of burst phenomena (such as the “pH-gush" which is clearly seen in the methyl- viologen reducing system). It should be stressed here that the light-induced pH rise in the medium (proton uptake) described in this paper has no direct bearing on the chemistry of the oxidation or reduction of dibromothymoquinone per se, as demon- strated by me the: that gramicidin abolishes the reversible pH rise but has no cfl‘ect on dibromothymoqtnnone reduction [l, 9} (see also Fig. l. traces C and D). In other words. the reduction of dibromothymoquinone consumes the same number of H‘ as are generated in the oxidation of water and therefore any pH changes observed iii the medium must represent the formation of proton gradients. Mechanism 0/ pro/Em truns/nc‘utimt associated it‘ll/l /’/lt’)(().\'_lMUN) / I electron transport The mechanism of the Photosystem ll “proton pump“ described above and its efficiency (ll*,’e’ --* 0.5) can be explained most easily if we assume that the mem- 134 522 brane-bound electron transport enzymes are arranged within the membrane in such a manner as to favor the vectoral movement of protons directly involved in electron transport. Such an anisotropy in the electron tiansport chain could result in a proton uptake with the observed stoichiometry (HU’e‘ ratio) according to two models: H20 —. 1/2 oz+2e' +2H*.t...d.i DBMIB-i—Ze'-Z—H+ ' H" (inside) ”- H20+DBMlB-+-H* (outside) —’ DBMIB”: -+ 1:2 02+DBMIBH2-T—H‘ (outside) (inside) or H20 41/2 02+2c- +H+ DBMIB-i-ZC‘ +2H+ H20+DBMlB+H* +H+ (inside) - (outside) (outside) —. DBMIBH: -* 1i?- OgrDBMlBHZ +H+ (outside) (inside) where DBMIB and DBMlBH2 represent the oxidized and reduced forms ofdibromo- thymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) respectively. The stoichiometric formation of the fully reduced dibromothymoquinone below pH 3 has in fact been confirmed (see Fig.3). It should be noted concerning the above models that, in theory, any intermediate situation between these two extremes is possible. One thing is clearly indicated by these formulations, however. That is, in any situation, a large portion (SO—10012;) of the protons produced in the oxidation of water must be released to the inside of the thylakoid, and asimilar and comple- mentary portion of the protons needed for the hydrogenation of dibromothymo- quinone must come from the outside (i.e. 50—100 ‘21, inversely related to the percen- tage of protons from water released inside). The exact point at which the protons eventually used in the reduction ofdibro- mothymoquinone enter into the electron transport chain is not known. However, there is kinetic evidence suggesting that dibromothymoquinone is primarily reduced via the plastoquinone pool [9], in line with the l‘act that its inhibitory site for electron transport is on the Photosystem 1 side of plastoquinone [24]. It seems reasonable to assume, therefore, that the protons which eventually are used in the reduction of dibromothymoquinone actually enter the electron transport chain at the level ofone of the plastoquinones, or even at the level of the primary electron acceptor for Photo- system II, which may also be a quinone-type substance [25 . However, we hasten to point out that it is still premature to assign the H *fe‘ ratio of 0.5, observed for this “isolated" Photosystem ll reaction, to the proton translocation efficiency intrinsic to the electrontransport pathway H30 -» Photosys- tem II -» plastoquinone since, for example, we have no clear idea of how the mem- brane permeating electron/hydrogen carrier dibromothymoquinone might alfect the anisotropy of that region of the membrane surrounding the plastoquinones. Some in- dication that the intrinsic value might be higher than 0.5 has been olfered by Rein- wald et al. [26], who showed a kinetic correspondence between the initial fast proton uptake ("pH gush") and plastoquinone reduction. They calculated the AH’U'M plastoquinone ratio (:22 H+,i'e‘) to be exactly I. On the other hand their interpreta- tion of the “pH gush“ may be questioned. As we have shown in Fig. 3e and f the “gush" as observed in this laboratory is not associated with an increased rate of elec- I35 523 tron transport. Furthermore there are also reports describing anomalously high H+/hv ratios (e.g. 5) [27, 28] at the onset of illumination. This too suggests a differ- ent mechanism of pH change rather than an increased rate of electros .ransport. Nevertheless, it seems quite possible that in the normal, complete electron transport system the efficiency of proton translocation (PF/e“) attributable to the H20 —v Photosystem II - plastoquinone portion of the chain may be somewhat higher than the values approaching 0.5 observed when this system is isolated from the complete chain. The relation between the accumulation of H + and the mechanism of phosphorylation at Coup/ing Site [I As shown in Table I, the efficiency of proton uptake (H+/e") associated with dibromothymoquinone reduction (i.e. associated with Coupling Site II) is 0.4—0.5 over a wide pH range (6.2-8.2). A striking parallel is found in the efficiency of phos- phorylation, P/e2 = 0.3—0.4 (ref. 9) which is also essentially pH-indepcndent over the same pH range. Furthermore, we have demonstrated unequivocally that concur- rent phosphorylation markedly depresses the steady-state extent of proton uptake in this system (see Fig.5 and Table II). These observations tempt us to postulate a coupling mechanism for Site II which involves the obligatory participation of H”. Indeed, the H*/e' ratio is so close to the P/e2 ratio that one is inclined to give con- siderable credence to the “chemiosmotic" coupling hypothesis of Mitchell [29]. The chemiosmotic hypothesis, as applied to chloroplast photophosphoryla- tion, postulates that the efllux of 2 protons through the membrane-bound ATP synthesizing enzyme produces 1 ATP (H",’ATP := 2). Various attempts have been made in recent years to determine this HUATP ratio experimentally. Junge et al. [30] obtained a value of 3 from their studies of flash-induced proton uptake and 5l5 nm absorbance changes. The same value was also obtained by Schroder et al. [31] who studied the kinetics of the dark proton elflux after the steady state. (A prototype of this latter experiment by Schwartz [32] gave a value of 2). Using some- what more direct measurements based on post-illumination phosphorylation (XE) experiments Izawa [33] found a value of 2.4. Thus, the experimentallydetermined H +/ATP ratios range between 2 and 3, which should be regarded as in good agreement with the hypothetical value of 2. In the Photosystem II reaction dealt with here, a approximation of the H+/ATP ratio may be made from the H*/e" and P/e2 values by assuming (a) that the H+/e‘ ratio determined from initial rates applies to the steady state, and (b) that all protons translocated are available for ATP formation. If we take the Site 11 ratio of H+/e‘ as 0.5 (see Results) and the Site II P/e2 as approximately 0.35 (see refs 3, 9), it follows that the requirement for protons in ATP formation (H‘/ATP) is approximately 2.9. While this is in good agreement with Mitchell‘s hypothetical value of 2 (ref. 29), it most likely represents an overestimation of the true H+/ATP ratio since there is probably a non-coupled. unspecific outward diffusion of protons com- peting with the ATP synthesizing pathway. Based on the data presented in refs 31 and 33, a rough estimation ofthe amount of this unspecific proton efflux may be made. Summarizing those published data, it can be reasonably concluded that the non- phosphorylating elflux constitutes 2040",; of the total proton cffiux (pH 8). The Fifi/ATP ratios corrected for this component would now fall very near 2. These considerations strongly implicate proton gradient formation as the mechanism of energy conservation at Coupling Site II. In addition, it seems likely that Site II can be identified as the water-oxidation reaction: the protons lost from water being discharged to the inside of the thylakoid. If this is so, the insensitivity of the electron transport rate to uncouplers of phosphorylation is easily understood [IO]; the electron transport results from an essentially irreversible photochemical reaction which is, as a consequence of its irreversibility, insensitive to concentration . of its proton product. ACKNOWLEDGEMENTS The authors would like to thank Dr N. E. Good for valuable discussions and for reading the manuscript. This work was supported by grants (0822657 and GB37959X) from the National Science Foundation, U.S.A. REFERENCES I Gould, J. M. and Izawa, S. (1973) Biochim. Biophys. Acta 314, 211—223 2 Gould, J. M., Izawa, S. and Good, N. E. (1973) Fed. Proc. 32, 632 3 Izawa, S., Gould, J. M., ()rt, D. R., Felker. P. and Good. N. E. (1973) Biochim. Biophys. Acta 305, ll9-l28 4 Trebst, A. and Reimer, S. (1973) Biochim. Biophys. Acta 305, I2 —139 5 Ouitrakul, R. and Izawa. S. (1973) Biochim. Biophys. Acta 305, 105—1 18 6 Avron. M. and Chance. 8. (I966) Brookhaven Symp. Biol. 19, 149—160 7 BOhme, H. and Cramer, W. A. (1972) Biochemistry 11, 1155-1160 8 Bradeen, D. A., Gould. J. M., Ort, D. R. and Winget. G. D. (1973) Plant Physiol. 52, 630-632 9 Could, I. M. and Izawa, S. (1973) Eur. J. Biochem. 37, 185-192 10 Could, J. M. and Ort, D. R. (1973) Biochim. Biophys. Acta 325, l57-It»6 Il Bohme, H., Reimer, S. and Trebst. A. (1971) .. Naturforsch. 266, 341-352 12 Trebst, A. (1971) in Proc. 2nd Int. Cong. 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U.S. 68, 2522- 2526 ' 24 Bohme. H. and Cramer, \V. A. (1971) FEES Lett. 15, 349-351 25 Stiehl. H. H. and Witt, H. T. (1969) Z. Naturforsch. 24b, 1588—1598 26 Reinwald, E., Stiehl. H. H. and Rumberg. It. (1968) Z. Naturforsch. 23b, 1616—1617 27 Dilley, R. A. and Vernon, L. P. (1967) Proc. Natl. Acad. Sci. U.S. 57, 395—399 28 Heath, R. L. (I972) Biochim. Biophys. Acta 256, 645—655 29 Mitchell, P. (1966) Biol. Rev. 41, 445402 30 Junge, W., Rumberg. 13. and Sehrdder, II. (1970) Eur. J. Biochem. 14, $95-$81 31 Schroder, H., Muhle, H. and Rumberg. B. (1971) in Proc. 2nd lnt. Congr. l’hotosyn. Res, Stresa (Forti. G.. Avron, M. and Melandri, A., eds) pp. ‘)l9-9?0. Dr W. Junk, N. V. Publishers, The Hague 32 Schwartz, M. (1968) Nature 219, 915-919 33 Izawa, S. (I970) Biochim. Biophys. Acta 223, 165—173 APPENDIX VII THE PHOSPHORYLATION SITE ASSOCIATED WITH THE OXIDATION OF EXDGENOUS DONORS OF ELECTRONS TO PHOTOSYSTEM I Reprinted from Biochimica et Biupliysim Acta, 387 (I975) l35—I48 (CD Elsevier Scientitic Publishing Company, Amsterdam — Printed in The Netherlands 88A 46893 THE PHOSPHORYLATION SITE ASSOCIATED WITH THE OXIDATION OF EXOGENOUS DONORS OF ELECTRONS TO PHOTOSYSTEM I J. MICHAEL GOULD" Department of Botany and Plant Pathology, Michigan State University, East Lansing, .‘Wich. 48824 (U.S./1.) (Received September 26th, 1974) SUMMARY 1. The Photosystem I-mediated transfer of electrons from diaminodurene, diaminotoluene and reduced 2,6-dichlorophenolindophenol to methylviologen is optimal at pH 8-S.5, where phosphorylation is also maximal. In the presence of superoxide dismutase, the efficiency of phosphorylation rises from g 0.1 at pH 6.5‘ to 0.6-0.7 at pH 8—8.5, regardless of the exogenous electron donor used. 2. The apparent Km (at pH 8.1) for diaminodurene is 6 - IO“ M and for dia- minotoluene is 1.2- IO" M.The concentrations ofdiaminodurene and diaminotoluene required to saturate the electron transport processes are > 2 mM and > 5 mM, respectively. At these higher electron'donor concentrations the rates of electron transport are markedly increased by phosphorylation (LS-fold) or by uncoupling conditions (2-fold). 3. Kinetic analysis of the transfer of electrons from reduced 2,6-dichloro- phenolindophenol (DCl PHz) to methylviologen indicates that two reactions with very different apparent Km values for DCIPH2 are involved. The rates of electron flux through both pathways are increased by phosphorylation or uncoupling conditions although only one ofthe pathways is coupled to ATP formation. No similar complica- tions are observed when diaminodurene or diaminotoluene serves as the electron donon 4. In the diaminodurene - methylviologen reaction, ATP formation and that part of the electron transport dependent upon ATP formation are partially inhibited by the energy transfer inhibitor HgClz. This partial inhibition of ATP formation rises to about 50 f’{, at less than 1 atom of mercury per 20 molecules of chlorophyll, then does not further increase until very much higher levels of mercury are added. Abbreviations: DCIPH,, reduced 2.6-dichlorophenolindophenol; HEPES. N-Z-hydroxy- ethylpiperazine-N’-ethanesulfonie acid; P,’cz, ratio of the number of molecules of ATP formed to the number of pairs ofelectrons transported; DCMU. 3-(3.4-dichlorophenyl)-l.I-dimethylurea. DBMIB. 2.S-dibromo-3-methyI-6-isopropyI-p-benzocuinone; HEPPS. N-2-hydroxyethylpiperazine-N-pro- panesull‘onic acid; EDAC. I-ethyl-3-(3-dimezhylaminopropyl)carbodiimide. * Present address: Section of Biochemistry, Molecular and Cell Biology, Wing Hall, Cornell University, Ithaca. NY. 14853. USA. I36 5. It is suggested that exogenous electron donors such as diaminodurene, diaminotoluene and DCIPH2 can substitute for an endogenous electron carrier in donating electrons to cytochromef via the mercury-sensitive coupling site (Site I) located on the main electron-transporting chain. If this is so, there would seem to be no reason for postulating yet another coupling site on a side branch of the electron transport chain in order to account for cyclic photophosphorylation. INTRODUCTION The transport of electrons from water to‘ conventional Hill reaction oxidants such as ferricyanide or methylviologen is coupled to ATP formation at two sites [1—4]. By the use of appropriate combinations of exogenous electron donors, electron acceptors and electron transport inhibitors it is possible to divide the complete electron transport process into two partial reactions each of which involves one ofthe two coupling sites [5]. This approach makes possible the study of segments of the electron transport chain and therefore the study of the functionally isolated coupling sites without physical disruption ofthe chloroplast lamellae by detergents or mechanical treatments. Through the use of these partial reactions it has been dis- covered that the two coupling sites in chloroplasts behave dill'ercntly in many respects [5, 13, 42]. Activities of the recently discovered coupling site [1—4] believed to be associated with water oxidation (see ref. 6), Coupling Site II, can best be separated from activi- ties of the well-known coupling site between plastoquinone and cytochromef [7, 8], Coupling Site I, by using lipophilic oxidants as acceptors of the electrons from Photosystem II. These “Class III“ acceptors (e.g. oxidized p-phenylenediamine) inter- cept electrons primarily at a point between the photosystems [9] (presumably before the plastoquinone pool [IO]). Thus when a Photosystem-I component ofthe reduction of these acceptors is eliminated by inactivating plastocyanin with KCN [l I ] or poly-L- lysine [12], or by blocking electron transport at plastoquinone with 2,5-dibromo-3- methyl-6-isopropyI-p-benzoquinone (DBMIB) [1-3], the resulting pure Photosystem II reaction includes only Coupling Site ll. Studies of Coupling Site II isolated in this manner have revealed the following characteristics: (a) Site II does not control the rate of electron transport since the rate is independent of phosphorylating or uncou- pling conditions (refs 9, IO and 13; but see refs 3 and 14); (b) Site II exhibits a charac- teristic phosphorylation efficiency (P/ez) ofO.3—O.4 [2-5, IO, II]which is independent of pH over the range 6-9 [5, IO]; (c) a light-driven reversible H*~uptake reaction is associated with the partial reaction [5]. The efficiency of this proton uptake (H‘/e) is 0.4—0.5 over the pH range 6—8.S [15]; finally (d) phosphorylation supported by Coupling Site II is not inhibited by the chloroplast energy transfer inhibitor HgCl2 [16, 17]. Similarly, it is possible to isolate Coupling Site I from Site II by inhibiting electron flow from Photosystem II with 3-(3,4-dichlorophenyI)-l,l-dimethylurea (DCM U) while using an appropriate exogenous source ofeIectrons for Photosystem I. In an earlier paper it was reported that the Photosystem l-dependent transport of electrons from reduced 2,6-dichlorophenolindophenol (DCIPHZ) to methylviologen utilizes Coupling Site I but not Coupling Site II [5]. Site I is probably a primary rate- I40 I37 determining step for the overall Hill reaction (H20 —o methylviologen) [7]. In fact. many of the characteristics of the DCIPH2 -. methylviologen and the water _. methylviologen reactions are similar. For instance. the rate of electron flux from DCIPH2 to methylviologen is markedly stimulated by phosphorylation or by uncou- pling conditions [5, 18-20] as is the overall Hill reaction. ATP formation supported by the transfer of electrons from DCIPI—I2 to methylviologen is partially inhibited by HgClz, as is phosphorylation in the overall Hill reaction [16. 17]. Moreover, the efliciency of phosphorylation (P/ez) is Strongly pH dependent in the DCIPH2 -> methylviologen reaction [5] as is the efficiency of phosphorylation in the overall Hill reaction. There are, however. some characteristics of the DCIPH2 _. methylviologen reaction which detract from its usefulness as a system for studying isolated Coupling Site I. For example, DCIPH2 donates electrons to the transport chain in at least two places. Most of the electrons from DCIPH2 are donated on the Photosystem 11 side ofcytochromef [21, 22]. This electron transport pathway is coupled to phosphoryla- tion (via Site I) and is sensitive to inhibition by KCN [5]. A second, smaller portion of the electrons from DCIPH2 are donated to P10“ via a KCN-insensitive pathway which is not coupled to phosphorylation [23, 24]. Furthermore, the proportion of the electron transport which is KCN-insensitive is a function of the physical state of the chloroplasts, being much greater in damaged or “leaky“ preparations [5]. In an effort to avoid some of the difficulties encountered with DCIPHz as electron donor, we have used other compounds which have long been known to be donors ofelectrons to Photosystem I (for a more complete discussion of Photosystem I reactions, see reviews by Trebst. contained in references 39 and 41). Several in- vestigators have reported that diaminodurene is an extremely efficient donor of electrons to Photosystem I, supporting very high rates of ATP formation [18, 25, 26], but the properties of the coupling site responsible for this phosphorylation have not previously been characterized, nor has the coupling site been definitely located. Trebst and coworkers have recently postulated that lipophilic exogenously added donors of electrons catalyze phosphorylation via an “artificial" coupling site formed when protons are released to the inner phase of the thylakoid upon oxidation of the donor by plastocyanin or P700 [39. 40]. In this paper we arrive at a slightly different conclusion: that the electron-transport pathway from diaminodurene (or from the chemically related compound diaminotoluene) to methylviologen very likely includes the coupling site located just before cytochrome 1' (Site I). If so, the high rates of electron transport and ATP formation associated with these partial reactions, as well as the absence of secondary electron pathways, make these systems superior to the DCIPH; -+ methylviologen reaction for the isolation anc.r study of Coupling Site I in chloroplasts. MATERIALS AND M ETHODS Chloroplasts were prepared from leaves of fresh market spinach (Spinacia oleracea L.) as described earlier [5]. However, for experiments with HgCl2 it was necessary to avoid the use of tricine buffer since this compound strongly complexes Hg“. Chloroplasts for use in these experiments were isolated in a similar manner, substituting N-2-hyt‘roxyethylpiperazine-N'-propanesulfonic acid (HEPPS)/NaOH 138 for the tricine or N—2-hydroxyethylpiperazine-i "'-ethanesulfonic acid (HEPES) where appropriate. In addition, EDTA was omitted from the grinding medium and bovine serum albumin was omitted from the suspension medium when HgCl2 was used. Electron transport was measured as the oxygen uptake resulting from the aerobic reoxidation of reduced methylviologen [18]. A membrane-covered Clark-type oxygen electrode was used. Reactions (2.0 ml Iinal volume) were run in thermostatted vessels at 19 “C. Orange actinic illumination (> 600 nm: > 5.00 kerg ° cm’2 - s“) was supplied by a SOO-W tungsten projector lamp. The beam was passed through a H round-bottom flask, containing a dilute CuSOd solution, which served both as a condensing lens and as a heat Iilter. ATP formation was determined as the 32Pi incorporation into ATP as described elsewhere [27]. Radioactivity was measured as Cerenkov radiation by the technique of Gould et al. [28]. Diaminodurene (Research Organic/Inorganic Chemical Corp.) and 2,5-di- aminotoluene (Aldrich) were dissolved in 0.1 M HCl, treated with Norit A, and recrystallized as the dihydrochlorides from concentrated HCI. Sodium 2,6-dichloro- phenolindophenol (Matheson) was dissolved in ethanol and liltered. Fresh stock solutions of diaminodurene, diaminotoluene and DCIP were made before each experiment. Diaminodurene and diaminotoluene were dissolved in 0.01 M HCI. Ethanolic solutions of DCIP were further diluted with I mM NazHCO3 so that the final concentration of ethanol in the reaction mixture did not exceed 0.5 ”4,. The concentration of the DCIP solution was determined from the absorbance at 600 nm using an extinction coethcient (a) of 2.1 - 10‘. Superoxide dismutase was prepared from fresh bovine erythrocytes by the procedure of McCord and Fridovich [29]. The specific activity. assayed as the inhibi- tion of cytochrome c reduction with xanthine oxidase. was > 3000 units mg protein. RESULTS Diaminodurene -’ methylviologen and diaminotoluene -> methylviologen Earlier studies of the Photosystem I-catalyzed transport of electrons from di- aminodurene to methylviologen indicated that very high rates of electron transport can occur. However, a tight coupling between electron transport and phosphoryla- tion was not apparent in these studies: the rate of electron transport being only slightly increased by phosphorylation or uncoupling conditions [18, 25]. Furthermore. the efficiency of phosphorylation (P/ez) was found to be rather low, usually about 0.3 [18]. In this paper we have re-examined the diaminodurene —. methylviologen reacrion in an effort to better characterize the relationship between electron transport and phosphorylation. Recently several workers have shown that the photooxidations of a number of exogenous electron donors can be associated with misleadingly high rates of oxygen uptake when methylviologen serves as the electron acceptor [30—33]. This is because significant (and variable) portions of the superoxide radicals ( - 02') generated by the aerobic reoxidation of the methylviologen radical react directly with the exogenous electron donor (AH;) and are reduced to peroxide (2 - 02‘ +AH2 —» 2 HO,‘+A). This leads to an exagerated rate of 02 uptake since some of the - 02‘. which normally dismutates to regenerate 1/2 of the consumed Oz (2 - Of—l-ZH" —. HZOZ-I-Oz), becomes trapped as additional H202: to the extent that superoxide I42 139 1.0 I r r r I to“ 0.5 °/°—° °‘~ a / 0L L l L I I 2500 i I I 1 T- 5 2000 at . [.5 Ta: - 1500 O. .— 4 W G} :5 lOOOl- ATP "‘ i o O c ; O— I 5 I. 3' 5500- -( Q, . 1 l 1 v 1 i 0 Or— O.1 0.2 0.3 0.4 0.5 s o 0 (mg/ml) Fig. I. Effect of bovine erythrocyte superoxide dismutase on the electron transport and phosphoryla- tion associated with the diaminodurene —’ methylviologen reaction. The 2.0-ml reaction mixture consisted of: 0.1 M sucrose. 2 mM MgC13, 50 mM tricine NaOH (pH 8.1). 0.75 mM ADP, 5 mM NazHJ’PO‘, 2.5/4M DCMU. 2.5 mM ascorbate. 2.5 mM diaminodurene, IOO/IM methylviologen. chlorOpIasts containing 5 pg chlorophyll and the indicated amount ol’superoxide dismutase (SOD). Note that although the oxygen uptake is lowered approximately 30 °,', by SOD. the rate of ATP formation is unaffected and therefore there is an increase in the apparent P/e; ratio from 0.3 10 0.5. For explanation see the text and ref. 30. reacts with the donor, the transport of a single electron results in the uptake of a whole molecule of oxygen. However, if the dismutation of- 02‘ is greatly enhanced with superoxide dismutase. the reaction with the electron donor is eliminated and the rate of 02 uptake becomes a reliable measure of the actual rate of electron transport, the uptake ofa molecule of oxygen representing the transport ofexactly two electrons (O, = e2 ). This is, ofcourse, only true ifthe chlorOplast preparation is free ofcatalase activity (which was the case with the chloroplasts used in this study). Fig. 1 shows the elTect of bovine erythrocyte superoxide dismutase on the Photosystem 1 reaction diaminodurene —+ methylviologen. As increasing amounts of the enzyme compete more effectively for - Of, the rate of 02 uptake is lowered by about 30 °/,,,. beyond which further addition of the enzyme has little effect. However. as might be expected, the rate of phosphorylation and therefore presumably the actual rate of electron transport is independent of superoxide dismutase. Because of this decrease in 02 uptake the apparent P/e2 (P/Oz) ratio increases from 0.3 to about 0.5. These data confirm the observation of f‘rt and Izawa [30], and indicate that accurate determinations of electron transport rates in the diaminodurene -. methylviologen reaction can only be obtained in the presence of a suitable amount of superoxide I43 140 1.0 1 Q~ 0.5;o’°—°_o——'o—“ Q o 1 1 4000‘- ' T— Aft)" .5 oqrom. O / _5 3000A . .__., ... 4 "F V +P / a ' P )— 0 ' i\ 3 2000i / ’o—°______O_. -i g 0 "6 [0’ E 0 2. 3 1000 / ATP "‘ .2 is ,""’°_°—'—o— 3 O :7 Ly 0 3 o.— t 1 O 2 4 6 DAD (mM) Fig. 2. Effect ofdiaminodurenc (DAD) concentration on the rate ot'electron transport and phospho- rylation in the diaminodurene —> methylviologen reaction. The reaction mixture was as described in Fig. 1. except that the diaminodurene concentration was varied as indicated. The concentration of superoxide dismutase was 0.4 mg/ml. When added. gramicidin was 4pg)ml. dismutase. Very similar effects ofsuperoxide dismutase were also observed when 5 m M diaminotoluene or 2,6-dichlorOphcnolindophenoI served as the electron donor. Since electron transport from diaminodurene (or diaminotoluene) to methyl- viologen exhibits many of the characteristics of the complete Hill reaction (H20 —. methylviologen), both reactions probably share the same rate-limiting step (i.e. Coupling Site I). At high diaminodurene concentrations (> 2 mM), the electron transport from diaminodurene to methylviologen is considerably accelerated by phosphorylation or to an even greater extent by uncoupling (Fig. 2). The failure of some earlier investigators to detect these large stimulations is perhaps due in part to the low concentrations of diaminodurene generally employed in the study of this reaction: typically < 0.5 mM. At these lower concentrations the donation ofelectrons by diaminodurene can be rate-limiting, and the effects of phosphorylation or uncou- pling on electron transport may be largely concealed. At the higher diaminodurene concentrations used here, however, the diaminodurene —> methylviologen reaction resembles very much both the DCIPH2 _. methylviologen and the H20 .. methyl- viologen reaction in its response to phosphorylating or uncoupling conditions, even though all rates are much higher in the diaminodurene system. The kinetics of the diaminodurene -» methylviologen reaction are shown by a double-reciprocal plot in Fig. 3. The rate-limitation imposed by the energy coupling mechanism is clearly revealed in this plot as departures from linearity. The data also indicate that this rate limitation can only be completely relaxed in the presence of an uncoupler. The reaction diaminotoluene —. methylviologen exhibits very similar charac- teristics to the diaminodurene -. methylviologen reaction. except (a) electron trans- 141 .0 UI I n h+qramicidin 14" l n A l '25 O - 25 5.0 7.5 _ 10.0 I/ DAD (mM) Fig. 3. Double-reciprocal plot showing the effect of diaminodurene concentration on electron transport in the diaminodurene —> methylviologen reaction. The data are replotted from Fig. 2. (Electron transport rates are in pequiv . h" per mg chlorophyll.) Note that. at very low diamino- durene concentrations. electron transport in the presence or absence of phosphate (P.) or the un- coupler gerniicidin is limited by the same rate-determining step. As the concentration ofdiaminodure- ne is increased. different rate~limitations become evident unless the reaction is uncoupled by gramici- din. port supported by diaminotoluene saturates at slightly higher concentrations (> 5 mM), and (b) the absolute rates of electron transport and phosphorylation are about 30 ‘34, lower under optimal conditions with diaminotoluene as the electron donor. Some of the characteristics of Photosystem I reactions catalyzed by diaminodurene, diaminotoluene and DCIPH2 are summarized in Table 1. TABLE I DIAMINODURENE. DIAMINOTOLUENE AND REDUCED 2.6-DICHLOROPHENOL- INDOPHENOL AS DONORS OF ELECTRONS TO PHOTOSYSTEM 1 Reactions were run as described in Fig. 2 when diaminodurene and diaminotoluene were the electron donors. or as in Fig. 5 when DCIPH, served as the eIeCtron donor. Electron donor Apparent K... Approximate concentration Approximate V (mM) required to saturate rates tpequiv — h" per mg (mM) chlorOphyll)* Diaminodurene 0.6 3 4000 Diaminotoluene 1.2 S 3000 DCIPH 0.03“ x 0.5.. : >0.6 > 1000 * Determined in the presence of Mag/ml gramicidin D. The rates are those obtained at the saturating or nearly saturating light intensities used in this study (see Methods). " High- and low-affinity components determined as described in ref. 34. See Fig. 5 and text for further explanation. 145 142 I0 1 1 I .~ I °’°‘°‘ \ 0.5 n a. / I a —A—./ o g l I # r 1 r #1 soool- ~ o/O~ T +Pl\/ z 0 u / _p g 2000" C) |\o‘ J ..~ 0 u: o'- O O/ t; // V' Z “ / {5’ 1000 ~ 2—3 “p\ - :61. 0’.‘-—.‘ 8 ./ .2 a -/ l g 0 -4—6/1 I J 1 6 7 8 9 pH Fig. 4. Effect of pH on the rate ofclectron transport and phosphorylation associated with the diami- nodurene —> methylviologen reaction. The reaction mixture was as described in Fig. 1, except that the concentration of diaminodurene was 3 mM. The concentration of superoxide dismutase was 0.37 mg/ml. The buffers (50 mM) employed were 2-(N-morpholino)oethanesulfonic acid/NaOH (triangles). HEPES/NaOH (squares) and tricine/NaOH (circles). Note that the efficiency ofphospho- rylation (P/ez) is strongly pH-dependent, being optimal at pH 8—9. and that a large stimulation of electron transport by phosphorylation is also seen at the higher pH values. These characteristics resemble very closely the characteristics of Coupling Site I in chloroplasts (see Introduction and ref. 5). Effects of pH on the diaminotoluene —+ methylviologen reaction are shown in Fig. 4. The rates of both electron transport and ATP formation are optimal at pH 8.5 or higher, with little or no phosphorylation occuring below pH 7. The P/ez ratio. therefore. shows the strong pH-dependence characteristic of Coupling Site I [5]. Indeed, the effects of pH on the H20 —» methylviologen, DCIPH2 -o methylviologen and diaminodurene —s methylviologen reactions are strikingly similar (compare Fig. 4 and ref. 5, Fig. I). DCIPH, -o methylviologen The widely used Photosystem I reaction transporting electrons from reduced DCIPH, to methylviologen probably utilizes the coupling site after plastoquinone and before cytochromef (i.e. Site 1) [21, 22]. However, it has been shown that the donation of electrons to Photosystem I by DCIPHZ has two components [5, 23, 24]. One component is coupled to phosphorylation (presumably via Site 1) and is sensitive to inactivation of plastocyanin by KCN treatment. The other component is not coupled to phosphorylation and is insensitive to KCN (ref. 5, see below). I46 I43 '0 T r #r o’°”f 9 ° a O 1 1 1 .5 ATP '2; l 5 ' -pl °/ s 412’ g / } 750~ ° 3 3e )5 / d. S ,/ E / .3 - o .3 SOOI-a -d| §2“/ ’6/ +P| 3 °/ +Pl ’Pt 8 , II/ /o a. [ = 0" .— “I o “4 § 250’ 0/40-1 ,I" I" ’7. +qrorp .2 I 'l I 0 8- x / m: 4.0/ .—— q, '.’o/°———-—'_'°- ,’ ‘0'. A ./ 2' 0&3" I 1 1 1 ---"'-’l’"’° t - o 02 0.4 0.6 '50 0 50 :00 DCIPHZ (mM) I/DCIPH2 (mM) % 2.0" .41! : o/ g 1.51- / E o +Pi § 10 - / 2 . '5 5 0.5 ~,°/ g 3;. +qrom. 4? _‘a—————6 4 j" 1 t 1 -lO 0 IO 20 30 1/0C1PH2 (mM) Fig. 5. :1. Effect of DCIPH, concentration on electron transport and phosphorylation in the DCIPH, -> methylviologen reaction. The reaction mixture (2.0 ml) contained 0.1 M sucrose. 2 mM MgCIb 50 mM t'icine/NaOH (pH 8.1), 0.75 mM ADP, 2.5/4M DCMU. lOOyM methylviologen. 2.5 mM ascorbate. 250 pg superoxide dismutase, chloroplasts containing 40 pg chlorophyll. and the indicated amount of DCIPHz. When added. NazHJ’POs (P.) was 5 mM; gramicidin was 4/ig/ml. Note that even at the lowest levels of DCIPH; tested, where the rates ofcouplcd electron flow are very low. a large stimulation of electron transport by gramicidin can be observed. b. Double-reciprocal plot of the data presented in a. Electron transport (circles. triangles) is in [tequiv- h" per mg chlorophyll; ATP formation (squares) is in ,umol ATP - h" per mg chlorOphyll. Note the biphasic nature of the plots. indicating that two different reactions with different affinities for DCIPH; are competing for DCIPH, as a substrate [34]. Note also that the rate of electron transport from DCIPH; to methyl- viologen is increased by phosphorylation and uncoupling conditions in both reactions. c. Replot of some ofthe data presented in b showing the component of the DCIPH; —> methylviologen reaction with the higher appartrt K... I47 144 The two-component nature of this reaction, also noted by Arntzen et al. [24], is illustrated in the double reciprocal plots in Figs 5b and 5c. The biphasic nature of the plots is characteristic of a system in which two reactions compete for the same substrate [34], DCIPH2 in this case. It is important to note. however. that both reac- tions appear to be dependent on the coupling state of the chloroplast, although only the reaction with the lower affinity for DCIPH; seems to support ATP formation (Fig. 5b); even at the lowest concentrations of DCIPH2 , where the high affinity, non- phosphorylating reaction predominates. significant stimulations of electron transport by uncouplers and by ADP+Pi are observed. Perhaps the access of the reduced indophenol to its electron donation sites within the lipid membrane is limited to some extent by the high energy state (proton gradient?) associated with the membrane. HgCl; inhibition HgCl; has been shown to act as a unique energy transfer inhibitor in chloroplasts at extremely low levels [35]. Concentrations of 50 nmol Hg:+ per mg chlorophyll or less inhibit ATP formation in the Hill reaction to a 50 "/,, inhibition plateau. It has recently been shown that HgCl2 inhibits phosphorylation associated with H20 —» methylviologen (Coupling Sites 11 and I) and DCIPH2 -+ methylviologen (Site I only) I T LO" .- N 3 05°\ a. 0—0—0 o— 3000*- " T o l I 1 E o 100 200 :00 a) HgCl2 (nmoles/mg ch11 1.5 5 20000 -l E < o_.__+P.l_ __ U) Q) ‘ ‘6 0 e U_ E ”Pg 3- lOOOr- "‘ o .2' J a b 0) 1 \._._. ATP .— o 1 1 O 100 200 300 HgCl2 (nmoles/mg chl) Fig. 6. Effect of the energy transfer inhibitor HgCl, on electron transport and phosphorylation associated with the diaminodurene -> methylviologen reaction. The reaction mixture was as in Fig. 1. except the buffer employed was 50 mM HEPPS/NaOH (pH 8.0). The concentration of superoxide d‘smutase was 0.37 mg/ml. ChlorOplasts were incubated with the indicated amount of HgCl; for 20 s in the dark before addition of the remainder ofthe reaction mixture. Note that ATP formation and that portion ofthe electron transport dependent upon phosphorylation are inhibited to a plateau of about 50 3:, by low concentrations of HgCl,. I48 145 but not phosphorylation associated with H30 —+ Class III acceptors (Site 11 only) [I 6]. The implication is that HgCl2 acts as a specific inhibitor of Coupling Site I. (The reason for the peculiar 50 ‘X, inhibition plateau is not at all understood.) Fig. 6 shows that the diaminodurene -» methylviologen reaction also includes the Hg-sensitive coupling site. The effects of HgCl; on the diaminodurene -+ methylviologen reaction pre- sented here are somewhat at variance with those reported by Bradeen and Winget [17]. They found that phosphorylation-coupled electron transport from diaminodurene to methylviologen was insensitive to low concentrations of HgCl2 (250 nmol/mg chloro- phyll) although phosphorylation itself was inhibited about 30",, by50-250 nmol HgCl; per mg chlorophyll. (Higher concentrations of mercury result in electron transport inhibition due to plastocyanin inactivation [36].) However, it should be pointed out that a small inhibition of a very fast reaction can often be difficult to detect when the electron transport is measured with the slow-responding, Clark-type oxygen electrode. DISCUSSION There are a variety of compelling reasons for believing that the primary path- way of electron transport from DCIPH2 to methylviologen includes the coupling site located between plastoquinone and cytochrome f. which we have called Site I. Izawa [21] and Larkum and Bonner [22]. on the basis of spectral evidence. and Neumann et al. [19]. on the basis of uncoupler studies. have suggested that DCIPH2 donates electrons to the transport chain at a point before a phospliorylation-dependent rate-limiting reaction on the Photosystem [1 side of cytochrome f. It has also been shown that the phosphorylation-coupled DCIPH2 —v methylviologen reaction resem- bles the coupled overall H20 —+ methylviologen reaction in its response to pH, to ADP-i-P,, to uncouplers, to HgClz, to other energy transfer inhibitors and to KCN [5]. It seems highly probable therefore that both reactions share the same rate-deter- mining coupling site. This conclusion is further strengthened by the fact that the sums of the phosphorylation efficiencies of the two partial reactions (H20 -+ Photosystem ll —. Class III acceptors, and DCIPH2 —» Photosystem I -. methylviologen are very close to the efficiencies observed for the overall H20 —. methylviologen reaction over the wide pH range 6—9 (see refs 5 and 42). Unfortunately the DCIPH2 —. methylviologen reaction is not altogether satisfactory for the study of Coupling Site I. As we have seen. the reaction is complex. The electron transport has a non-phosphorylating component which utilizes a different pathway and the magnitude of this component varies with the state of the chlorOplast membrane (see Fig. 5 and refs. 5. 22-24). The results presented in this paper seem to indicate that the photooxidations of diaminodurene or diaminotoluene are partial reactions of the electron transport chain which also involve Coupling Site 1. Again the effects of pH, of ADP+P,, of uncou- plers, of HgCl; and of KCN are similar in the diaminodurene (or diaminotoluene) -v methylviologen, in the DCIPH; —» methylviologen and in the overall H20 —> methylviologen reactions. Therefore. the criteria of Site I involvement applied to the DCIPH; reaction may also be applied to the diaminodurene (or diaminotoluene) .. methylviologen reactions. This conclusion is strongly supported by the recent finding I49 I46 DCIPH2 DAD, Class III DAT . acceptors l ocuu 1" DBMIB EDAC KCN Hzo—-Ps 111]» --PO Went-4°C —¢Ps 1—-—~MV ~ ma, ~ SlTE II SITE I Fig. 7. A scheme for electron transport pathways in isolated chloroplasts showing the two sites of energy transduction (~). PS 11. Photosystem 11; PS I. Photosystem 1; PO, plastoquinone; cyt f. cytochromef; PC. plastocyanin: DAD. diaminodurene; DAT. diaminotoluene; MV, methylviologen: Class III acceptors include lipophilic strong oxidants such as oxidized p-phenylenediamincs or [to benzoquinones. Note that in this scheme diaminodurene. diaminotoluene and DCIPH, donate electrons directly to the main electron transport chain at a point before the rate-determining Coupling Site I. which also limits the rate ofelectron transport from H20 to methylviologen. NOte also the two component pathways ofthe DCIPH, -—> methylviologen reaction. one KCNsensitive and one KCN- insensitive [5]. For further explanation see refs 2—6. 8—11. 16. 23 and 37. of McCarty [37] that the electron transport inhibitor l-ethyl-3-(3-dimethylamino- propyl)carbodiimide (EDAC), which apparently blocks electron flow from plasto- quinone to cytochrome/at some point after the DBMIB inhibition site. inhibits the transport of electrons from diaminodurene to methylviologen. This suggests that, while the chemiosmotic model for Photosystem I phosphorylation may be basically correct, the actual acceptor of electrons from diaminodurene is not plastocyanin (as suggested by Trebst [39, 40]) but cytochrome f, as is clearly the case with the coupled DCIPH; -> methylviologen reaction [21, 22]. This is consistent also with the fact that no energy control over electron transport can be detected between cytochrome fand NADP+ [7, 8]. A scheme summarizing these observations and conclusions is presented in Fig. 7. One important implication of the results summarized above concerns the probable location of the coupling site associated with Photosystem 1 cyclic phos- phorylation reactions such as those catalyzed by pyocyanine. diaminodurene/oxidizcd diaminodurene, or DCIPHz/DCIP. It has long been postulated that cyclic photo- phosphorylation is associated with a separate coupling site not on the main electron transport chain (for a review, see ref. 38). In light of the evidence presented here it seems more likely that the reduced form of the cycling cofactor (i.e. DCIPHz. diaminodurene or reduced pyocyanine) donates electrons directly to the main electron transport chain at a point before Coupling Site I (i.e. before cytochromef, but see ref. 40) while the oxidized form of the cycling cofactor (DCIP, oxidized diamino- durene or pyocyanine) accepts electrons from Photosystem 1. Trebst and eo-workers have recently presented a similar view [4, 39, 40]. Such schemes eliminate altogether the need to postulate a special "cyclic“ coupling site associated with a separate elec- tron-transport side chain. Instead these schemes have cyclic phosphorylation resulting from the proton translocating properties ofthe exogenously added lipophilic electron I47 donor: i.e. the donor is reduced (consuming protons) on the outside and oxidized (releasing protons) on the inside ofthe thylakoid, thereby generating a transmembrane proton gradient which may drive phosphorylation. The scheme presented in Fig. 7 is more specific in that cytochromefis suggested to be the acceptor ofelectrons from the exogenous (artificial) electron donor (e.g. diaminodurene) as well as the endoge- nous (natural) electron donor plastoquinone. Studies with electron transport inhibitors also lend support to the scheme. Ouitrakul and Izawa [l l ] found that KCN inactiva- tion of plastocyanin inhibited cyclic phosphorylation catalyzed by DCIPHz/DCIP, diaminodurene/oxidized diaminodurene, pyocyanine and low concentrations of N- methylphenazonium methosulfate (PMS)*, showing that cyclic electron transport shares at least this carrier with non-cyclic electron transport. Furthermore, McCarty [37] has shown that pyocyanine-mediated cyclic phosphorylation is inhibited by EDAC. Thus it seems clear that cytochrome f (and therefore probably Coupling Site I) is involved in the cyclic reaction [37]. If we are to equate the site of cyclic photophosphorylation and other Photo- system l-mediated phosphorylation reactions with the rate-limiting Coupling Site I of the non-cyclic electron transport system, a problem of relative rates arises, since the Photosystem l photophosphorylation can be several times faster than the non-cyclic photophosphorylation associated with the Hill reaction. This apparent conflict, how- ever, may be easily explained in terms of the differences in the concentrations and reactivities of the different electron donors (e.g. diaminodurene vs. natural electron donor) at the common site of oxidation, where energy conservation takes place. Electron transport rates of all ofthese reactions can be under comparable degrees of energy control, regardless of the electron flux, since the steady-state level of the high- energy intermediate or state (proton gradient?) and therefore the back pressure it imposes on electron transport through the coupling site [13], may well increase or decrease in approximate proportion to the level of electron flux. The result would be a similar degree ofdependence ofelectron flow on phosphorylation and on uncoupling regardless of the absolute level of electron flux. ACKNOWLEDGEM ENTS The author would like to thank D. R. Ort, S. Izawa and N. E. Good for many helpful discussions. I would also like to thank Dr R. E. McCarty for making available a manuscript on EDAC prior to publication. D. R. O. and Ms S. Perry provided invaluable assistance in the isolation of superoxide dismutase. This work was sup- ported by a grant (G837959X) from the National Science Foundation, U.S.A., to Drs N. E. Good and S. Izawa. REFERENCES l Gould, J. M., Izawa, S., and Good. N. E. (1973) Fed. Proc. 32. 632 2 Izawa. S., Gould, J. M., Ort, D. R., Felker, P. and Good. N. E. (1973) Biochim. Biophys. Acta 305, 119-128 * Cyclic phosphorylation reactions catalyzed by PMS are complex. the properties apparently varying with the PMS concentration. See refs ll. 27 and 23. ' l9 20 ‘l '31 -b 23 24 25 26 27 28 29 30 3| 32 33 34 35 36 37 38 39 40 4| 42 )5) Trebst. A. and Reimer. 5. (I973) Biochim. Biophys. Acta 305. l29—I39 Trebst. A. and Reimer. S. (I973) Z. Naturforsch. 28c. 7I0 f'l6 Gou1d,]. M. and Izawa. S. (1973) Biochim. Biophys. Acta 3M. 2! l—223 Izawa. S. and Ort. D. R. (I974) Biochim. Biophys. Acta 357. l27-I43 Avron. M. and Chance. 8. (I966) Brookhaven Symp. Biol. I9. l49-I60 BOhme, H. and Cramer. W. A. (I972) Biochemistry II. llSS-I 160 Saha. S.. Ouitrakul. R.. Izawa. S. and Good. N. E. (I97I) J. Biol. Chem. 246. 3204-3209 Gould. J. M. and Izawa. S. (I973) Eur. J. Biochem. 37. ISS—l92 Ouitrakul. R. and Izawa. S. (I973) Biochim. Biophys. Acta 305. l05-I I8 Ort. D. R.. Izawa. 3.. Good. N. E. and Krogmann. D. W. (I973) FEBS Lett. 3l. lI9-l22 Gould. J. M. and Ort. D. R. (I973) Biochim. Biophys. Acta 325. 157-166 Heathcote. P. and Hall. D. 0. (I974) Biochem. Biophys. Res. Commun. 56. 767-774 Gould. J. M. and Izawa. S. (1974) Biochim. BiOphys. Acta 333. 509-524 Bradeen. D. A.. Gould. J. M.. Ort. D. R. and Winget. G. D. (I973) Plant Physiol. 52. 680-682 Bradeen. D. A. and Winget. G. D. (I974) Biochim. Biophys. Acta 333. 33l—342 Izawa. S.. Connolly, T. N., Winget. G. D. and Good. N. E. (I966) Brookhaven Symp. Biol. l9. l69-I84 Neumann. J.. Arntzen. C. J. and Dilley. R. A. (I97!) Biochemistry IO. 866-873 Strotmann. H. and von Cosslcn. C. (I972) Z. Naturforsch. 24 . ”RX-4598 Izawa. S. (I968) in Comparative Biochemistry and Biophysics of Photosynthesis (Shibata. K.. Takamiya, A.. Jagendorf. A. T. and Fuller. R. C.. eds). pp. l40-l-‘17. University Park Press. State College. Pa. Larkum. A. W. D. and Bonner. W. D. (I972) Biochim. Biophys. Acta 267. l49-I59 Izawa. S., Kraayenhof, R.. Ruuge. E. K. and Devault. D. (I973) Biochim. Biophys. Acta 3l4. 328-339 Arntzen. C. J., Neumann. J. and Dilley. R. A. (I97I) Bioenergetics 2. 78-83 Trebst. A. and Pistorius. E. (I965) Z. Naturforsch. 20b. l43-I47 Hauska. G. A.. McCarty. R. E. and Racker. E. (I970) Biochim. Biophys. Acta I97. 206-218 Saha. S. and Good. N. E. (I970) J. Biol. Chem. 245. 50l7-502l Gould, J. M.. Cather. R. and Winget. G. D. ([972) Anal. Biochem. 50. $40-$48 McCord. J. M. and Fridovich. l. (I969) J. Biol. Chem. 244, 6049-6055 Ort. D. R. and Izawa. S. (I974) Plant Physiol. 53. 370—376 Allen. J. F. and Hall. D. 0. (I973) Biochem. Biophys. Res. Commun. 52. 856-862 Elstner. E. F. and Kramer. R. (I973) Biochim. Biophys. Acta 3I4. 340—353 Epcl. B. L. and Neumann. J. (I973) Biochim. Biophys. Acta 325. 520-529 Dixon. M. and Webb. E. C. (I964) in Enzymes. 2nd edn. pp. 87-90. Academic Press. New York lzawa. S. and Good. N. E. (I969) Prog. Photosynth. Res. lll. l288—l29x Kimimura. M. and Katoh. S. (I972) Biochim. Biophys. Acta 283. 268-27 McCarty. R. E. ((974) Arch. Biochem. Biophys. I6}. 93-99 Avron. M. and Neumann. J. (I968) Annu. Rev. Plant Physiol. )9, l37-I66 Trebst. A. (I974) Annu. Rev. Plant Physiol. 25. 423-458 Hauska. 0.. Reimer. S. and Trebst. A. (I974) Biochim. Biophys. Acta 357. l-l3 Trebst. A. (1972) Methods Enzymol. 24b. l46—I6S Izawa. S., Ort, D. R.. Gould. J. M. and Good. N. E. (I974) Proceedings ofthe Third International Congress on Photosynthesis. Rehovoth. in the press APPENDIX VIII ELECTRON TRANSPORT REACTIONS, ENERGY CONSERVATION REACTIONS AND PHOSPHORYLATION IN CHLOROPLASTS .o ’n .o ,.o . J a g .. I o u y. "(It .e.'..7.1‘ i‘ .n; .I..7‘.. M. AVRON, Fr r,. 1? l5 vou' / ‘ 'f‘,v ‘9). .c g -, ere. “wan ¢.vss:n:F‘otrs , September 2-6. 1976, Keizmann Institute of Science, Rchovot, Israel Elsevier Scientific Publishing Company, Amsterdam, The chhcrlands, 1974 Z'ECTROH 737132337 REACTISNS, E 390? V U‘FBYATICH R 5072233 ‘\'v :‘-;r-4" ‘.:‘I"v «v‘vn‘.o for -.v’f ‘0” . .rv 11.. .e.»...v. 4!‘.--"' _ q .u i . AV. 3. :awa, 3. R. art, J. W. Goa-d, and H. . good Department of Botany and Plant Pa‘bolovv, Vieiiran State UniVerslty East Lans;:m, Michigan ~37?4 _. 3. A.) I. Lseation o: the Sites of Phosrhor"latian This part of the paper deals with the use of a variety of elec- tron donors, electron acceptors, and specific electron ‘ransrort in— hibitors to isolate quite different ocilation—“eduetlon reaction“ ‘ N‘. " .- -' ' s . ~. ,— .- a responsible :or 6-? tormat-on 1n chloroplasts. :igurz l summar-:es the partial reactions NHZOH DCMU ' I O I Site It i ; i P. I e H O-—:-—OPSII - 2 ; I‘d iCatechoI thus investiq DBMIB l I I o "Ham—~— er 4 '54:“ ed. KCN Sitel DA04---:----- L—_.'_..Ps I ~ "! I0. _.--_--fig-_--- PC'PSl—’MV —L-»M\/ >PS I —>MV ‘ 1 r‘ O-l ‘. . ‘7‘ ‘~ ‘2 .- Figure l. Chloroplast reactions currently anal.aole :.r the study of photosynthetic electron transrcr“ and rho"'*n“yltti.n ‘ m - w T T . - >- -. .- Firure -a. .he ‘cneme shows the overa l electron ransport I ‘ J 'V ‘ n‘ ‘ R |v .V" ‘ system, the probable oosltlons of tne tran-oort o-oem- it-l;ht:, and the apparent location of the two sites of e'er~" ecnservation. Let ‘ a v 4 .1 .' -- - us first review tue nature 0. tne inn-bit-ons ind-eated by :ne - '- 1.. - - ,. "'\ .--°- s -' . ".°-‘:. :-‘. a... t.‘ abbreVLat-ons used. UMJ, alaninedureno, -_m-d, .. -; : oro—;---'3.- p, ‘7' ' .' ‘-.‘v -. l -“ ‘t- 6-isopropyl-o-benchuinone; ItiRh; realee: to 2 o .,t-a-:n_3rr- < ‘ ~ - ’.001' '3 '- i -"'r‘ 1 \ \ ~ “ I ‘ ‘_ . fi..\ phenolindophenol, D; t, ;-tj,d-oi:hlorag. vi -l,_-c-.et:vlur~a, on N ' .w ..-' . P“! - -' " . ......-. .‘ , . .. . ‘ :,5-dimetnyt-p-benzoqu-nsne, -AFD, “,J,H3I - etza . ”J —s-))on.-err— diamine. IS4 450 IZAKA, nRT. COULD AND GOOD vertical barn across the tranntort chair. Hydroxy' been knowr to an inhibitor of rnotosyntfiosls and :h» More reee.tlv it 335 been recofirizejtntt thi: am‘h- water-0x1 lain; system il-g. as do tth r an‘nr: iu‘ Tue arcat advantaga OI hvurox" e"‘re-r“e““'eut is 7 carried out in such a manner as to totally and srec water oxidation without adversely affecting the no: chloroplasts to onesrno"ylate AD? {5). Hydr‘xrlaxl plasts are nirh’t ac'i‘v in 'hr Photcc"st a fl-z-ve 1 | A _‘ _1 _ 1. l,‘ .‘ ' _‘ .Ir I..-..-- 01 $0030 QXOCGnuu: electron aonots unttn it: -.sdt- DCMU is the standard specific lnnibitcr of Photogr; Dibromoisorroiylbcnzoauinone (Dfifif?) U L]. 7V (O pd d ..- ,- ‘4 (. a ha y \M ‘2 I \ ‘ h 0 J . . .' .‘ r e. p f. , . .. ‘ . ,‘, . o..- .. , ‘ J. L. SSOCir‘. 133.! ‘6] ‘ -5; .l Ybr‘-.'.ie 40'.'.1‘IILII.’.I \i'l atak r’I-gy ..i‘-\."l the reoxidation of rogue d plastoqu none .-.2' Eu; “ 9" a, h . P " \ .. 6-» "’ ‘_‘._..-, transter Cl elec-r ns _rom Bhotesystcn -t to .H3:» for of electron: between tne two nnotasy tom: 1; a? J c. 6‘ .. ‘ 1 , ,_ I,‘ 5 . r ‘ s v I ( prior treatment 0. the chloroplasts w-tn '.a J). . - r A .- j . . . , . . nuubithwi resalts from tnacttvatton o: clasto .an-n ('3 (3 of the consequences of th 510 k are suite dIRFaren sequences of US$73 inhibition (see selow‘. The ear the electron transport system made available by tne hibitors are shown in finuro ; (0,0 and i). t." T'- " . i ‘ '- a. - ‘, ..,6- ‘, ..| . . TLITG .J . it I.‘....'T'!l):‘ I" 02 eaogvsno Ji. t,',_‘_ ,Y'DII J Jilt- minodurere (OLD) ari rwduc d lnnonnwnol 'i31?d-l ca. 6— p “friih‘fi- I "‘_,'\,_ .y.-.‘p;+ ‘_,‘ n ‘. ‘."‘: » . Ilyit .“ A thLl.’J Q .‘.‘m .. 2.3;; L. .-l\.;r I . ' .4 ("1‘ . I i "x... J ..‘."‘ “.‘. J .i . .1 a. I . .~'. . .- ....,-°.: ‘- '. hese tamlllar reaction: a?“ insensitive .2 n/.roat DBMIB (7). In t*r nast t? O 3 (D Q) (.1 C "S (D L: O O ,_. L. 3 J t 4 a; ‘7 '3 (V C '3 L.) ('1 k) 1‘) D I; (I) (I -) ‘3 —. v '. . _. ..: - , . . .. xidation OI reduced ew‘rv-.-o- 5‘“ "L' '">n axeuu' ‘o— ' A r .. ,- ‘ 2.. electron donor w-,n C'e v2rs .scn:t 2 it :3: a- 8.? _J I I I I: v be overes imaged (l- - form of the donor to accept electrons. superced1n‘ ed, can result in a hidden evctiz vlwcfiran tiow wit that the electron transport ma? be unierostimatnd. blem is oarticularl acute when :ne re-at:v~lv ice“ ferredoxin acceptor svstem '= us; time the only r:-iablc procedure for mrngariur tie datiOns of exogenous olectror loner: involves thx=i acceptor such as nothvlvlolcf.n unier 1~ro:i: c4xu; ascorbate to Prevent the accumalotion 2? the (Vii-m (‘J (4‘ e f (I J (“J (1 gr (‘Y ("Y r" m t) p A. (1’ IZAKA. donor, an tn» use 0. large thuac these txvxxrlt‘orc cm": : phosphorylsticn 3012::On can r1’lc 0” AT. sclezu e‘ forne measured in t.:s manner is c electron donor used 1:3,15 re:;NJnsilJle for‘ the gxhcc'fl‘wr since this seems to be t'e : reduction of cytochrome C (2 good reasons for be-icv‘n¢ “ so-called "t”Clic okcsrrory' as LAD or py’rycnine. In an cyclic, are inhibite” if 91; polv-‘-lysine (if). FLJAre 1c. Lirophilic (DFKQE, \r'idix ‘4 gz-rznsnyl-‘cdi (D‘D ) can interceht oi ctr OX ‘ Thus the electron trnn33ort if firm with K transport, tion w ed the wa S t he h ? VN IB is <9) 1th sitc an O L. SCCCH’ .‘N . sys em II. Figure 13. chloroplasts ctn oxidize a number on Pho trons ferroc not in to phosphorylation - b the are onerati on the and 31 be one the ne standi (‘O- «J ‘4 tosy can be yanide, the _ nature te 11 r xt n S CH of which f fi“ -‘ ‘ .meu'f . (sec r\ of oresence of i I ‘ fiffifltvy J -t .9 1" CRT, Ghent r» l .o ’4. 7" .‘ Ll H ’x Y I\ i (T p v .A. U ‘ ore " h A ~ ‘ l I C‘ J ‘ rub ". lak)‘ L3” 9. v $7 or 3"": F 04,143 ‘r ohospnory n D |—h (V 1&- 5 ‘ v A)?“ CDC") r. ,. ,.) P. I. f‘ F? '7‘ ,4- i.‘ 45.3- ,1- I -J.l- ‘0 ' can! (i ‘ 9 1 V \f ‘. u. ; b1 '— l 156 IZAWA, CRT, GOULD AND GOOD I 1500? E5 0 o 01 C) (3 pequiv/hrwng chl on I HzaneCy "\J O O Fivure 2. rons PDox) hotosyste DBMIB and tors. transport of Photosyste both photo t x ? II. (a) A gre electron t formation "chemiosmo (30) and, confirmed considerable of the water (up; about 0.4 30 60 9O KCN-Treatment (min) .t “14".“ A J1. Reduction of a lipophil ‘ o- t "3". Curr: .I to - 4.“. .1- “ ,-. .. m 3 4 n - : 1 . -, . - :rom mater. .he reuuct-on o- ox;d.zcd n-rh:n;-enul;»n no -. ,~ -. 1 - ., .- , . 4, , . _. ,- ‘ has ZHO comPOLents, a large corhoneat an-:h deuenl- JL-: on m II and Is therefore insenrlti"e to "CV-treatmrwtt or “ — . ' ; R -" ~. , '2 ,1 . ...‘ u ‘ . . ° v 3 .z a smaller component ~h_cr .s 2; x-natuo Ly those -nr t.- Xote that the large Photosystem I: ccmocncnt c. the -Lcctron ‘ -. - _ ~ : u' A ~ -. ‘ ‘° . . I '7 ‘ supports nnosunorylation wt.n the oflmU e::;-i'rc" ar/eq, ‘ —. ». ‘ 1 ' ‘ : .t . z ‘ . . .- .. Gt. regard-ess of whlch 1nn-t-tor -u Jsei .o n m-na:e .ne m I contribution. Ferrtcranicc reduction, which invc17es ., .. ; . 1' .- - . J . ..‘ . ‘ .Y : .‘ systems -n those cn‘aropliy‘s, ': .nuwn ’C" :orrar_son. ' . :- ‘,- 1 ~ ‘ f“ - s ‘ '1 Cnaracterlzatlon o: the “itzs of rnosprozylat-on ‘ l » \ ..' . "-. - f - . l.‘Y-.,. "r Proton proouct-on and phosgnor;-atlon 4t c-.c l. ‘ ‘ . 4 -' . . . , '. . ; .’ ¢ ._ - ‘l . at deal 0: evlnence has locumulatcc which In.‘rrc-ntc3 . t. 0 ° F . ~ . 1 - . z - c... ' nu") ran: rort, the lcrmatlon o. Y.f,’-i!‘0.~ft-H -r.n :‘at-i-ent..,lr .: 11-: 4 ‘ . ‘ - 5.. v n -,_ - . . .K _. ‘ In chloroplasts. Aacn o- tnis eulsuncc stucrts the : '0 ‘.~ I .‘ -° .0 x - ‘ ‘ ‘ tlc cdolomitlon o: ohosthor3-at-on orctc.cc 'y -cnrl- ‘ 3 6-4 " J o- 1 V _‘ ' -. -ndecc, sore predic.-ons 01 --che.l s tneory ha.: o:cn : 2 v. 1 ‘ t-.-r - '-- "c -' "’ ‘ " ' by exper-ments so beaut_lul-y uni: the tnn-:; nae ~a_ned "' . ' 1 ‘6- . - . t-Ll ~. A ‘44 popularit". riglre 3 ll-astrates o.e “res.rcl w “n ngs 4 ..‘ 1 ‘ « .‘ ,-. - ‘.«, chem-csmot-c meccanlsm in cn-oroolasts whet tor. are o-‘.l;_ng . K V ‘- a _ x , _. . -., er) or eyogcnous donors _lowcr, (51, ‘or a “lecw sec ;2, 157 IZAWA, ORT. CQL'LD AND CW5!) A53 1 PS” i e I~p PQH‘z-Jvf' PC'PS' i + \H+ PSH PQHZ- CotechoF—_ DA 0 \ \ *- H Figure J. A conventional chemios".ctic interpretatic n o: phos- phorylation in chloroplasts. The upper figure snous a s-rment of a thylakoi d membrane oxioizing water. The lower segnont ).:wc a segment oxidiz ing exogenous electron dorors. The essential features of this model arsr a) Ihot osys‘em I ox jat rs and Photosystem II n J.- » 4 oxidations ooth result in the accumula: of protzns insiia tne thyla koii. b) These protons are extruded thrornfi the o!u'3' in such a manner as to acne rate AT? - f“ I C‘ '3 According to the moiel hydrogen donors are oxidized on or near the inside of the membrane and as a result hydrogen ions are ‘3 k. H il 1‘ D x L. into the inner space of the thyla.oid. This is believefi to happen when water is oxidized by Photo S‘sten II (3?-35) and again whensore intermediate hydrogen carrier such as plasttquinone is oxidized by Photosystem I via cytochrome f {33). The inner protons then somehow generate ATP while diffusing out of the thvlakoid via the couplinv factor (30). The two proton-producing reactions thus constitutcthn two ”sites” of phosphorylation descrio The concept of inter.al proton produ mediate step in ATP formation has received vo y strong support from experiments with chloroplasts. As Hauska et al.{?“) have 90' out, the fact that the oxidation o? DAD 5y Photosystem I supp phosphorylation while the oxidation of THFT noes not ma" be a con— sequence of the fact that DAD oxidation result in nydror en ion pro- 1‘) duction whereas the res oval of an electron from T??? produces onlx the free radical. We have now shown that a similar correlation be- kg- tween proton rele ase and AT? formation is observed wnén exonenOUs 158 5!. IT’..\'»-.'.\, ORT, GOULD 33"." 1300?) electron donors the o llizel by Photonystcm 7T. Efflur~ 4 :~L; re: the c'ication of c- wchol ty ul‘“CNYlQT‘V -CI'“1'J v:-;:unll;‘ ”:tn the oxidation of fvPTOCyfihiifi by the sane onloro: .s ‘. H‘vn “‘3- chol ‘s OXLJlZ'“ the :f“‘*1? n" of n415'hor ‘ati n to aln c: Cotechol Ferrocy- 1'. o- ‘ "'MV l.O$’-‘- --.------ —+MV l.O----------- P/82 (15¢tfltrfiib0~ 150*- l J i O O 20 4O 60 les/hr- mg chl .0 ow 2 AT: ° N ‘ 50b ET(xI/2\ o " Moot gyow—‘o- . O/ °~o~o- 5 /// “\ELT.(x!/2) /// .3 ,9 25- /0 O—o—o— " g 50"' + O ‘//°’f: °- P L 1 0| I l l L O 0.5 LO 0 20 4O 60 Cotechol (mM) Ferrocyonide (mM) m 93, . ll ' 7 ‘ ' ' Fieire 4. .he oxloatinn of :atecno- aha terrocvnh;de tz'nvi““?v- ‘ _ .1 , a A . u - “ *‘ ‘ . -amine-treated chlorouiastn xl‘n DHIHVIVlH“‘Dr ~~ n vot~“n i“‘““’0“ ‘ . “..‘, - t-- .--' ..‘; 4...»- 45 .A __.l. -s (“a \" ‘ I. H . .0 , . \ ug‘ \ n » - .heue reactions are Lhncbltei by Fig} ZLQ fnzr~1,re r;nu-re .‘ota- -. T ‘ -\-l — . ‘ ' ' ‘ . alibi-1r)!“ L1 . :“1".'ac .4} '1“ t? r‘ (I? 2 :'U> ‘ .‘V‘l \“ “f‘.*'4‘."-\‘ -. v V“-1:‘l". . .~‘O " y , $19.— ,, ..—. _~.—- v .vu. - - ~». . -'- -‘ i v», -.'. .,~ . 'A v l ‘- . I ’ ‘1 p o I U lation with an e:LICIencw ance! 7HIAC.C s Lo-~ ““‘ 1r: "2 I' 'flhereas txv~taxijatixuz t f:r":t'u.lau so i”" tiw':tn~r“ " ui'vi‘h ‘ «.‘-. . .L . . . N , . h \ w ' ‘. : , l .- ‘_ , , .- c. .' . .- .. . - . . . ' - ’ " ‘ Lut e.fic-cncy WH_Ch is c aricter-u,l: t; .he cgcr:;-on if wipe - alone. ‘ . r- 4" ‘ l ‘ .. . . . the same as wnen Nate? is ox;d-ztd and -her~fore hotr Jite I ani (2“ T7 -’- "‘ ‘ mite ll must be operatilw -3). On tie other hzn:} 5P6“ ”arrtc'i- 1 - t n \: : l . .. . _ ‘ ' ' ‘ nlde s oxidize: the e.f “-ULC/ o. t'usn* "2' "e" ‘ ;'oreu 1) ‘ .._~d~o .I‘ A -- J r! n - u about halt a-thou,o fwrr0t"an;:c 1t th~ 13n,_ntr“:i~n3 use: has w unoowpl'ht ef'unt (“0) *n“ ' ..‘ ‘hr ~ *t' t ' ‘ .l_ A... 'y A) a C, I I - U‘: ‘4 4L2'- prilJ~L .lv)’ I'd -.’-CY“. .A‘s.~."’:_'.l.zl’1 :i‘tr: ‘- p I ‘ 1 fl '_ I the transnort o- electror‘ frat Larrooyaridt t) nethyli;7l‘ren '13 8‘]. Op '- ‘3 "r0 3 “4 .0 W" {‘“n rl “' "'*:‘ ‘ - . the p ptr,-; t. --- uiottnor'-. -cn 1" n‘nw o: ' ‘ h‘ o (w 7’ .P a- ‘ - . ’ . nrOpe ties of .1: i; rrosnh:rvlat-on e1; *ea gist f;rure t ::1ou). u when ferrocyanide “Pure eleCtron ionorl sibatitutns for water :m-for ' " ‘ 4 .. T 4 -. ‘~ , . .. ,_. . catecnoi (hydrogen donors). -nc-ien‘al-5, ‘*~ ”:rt .Uzt tniu re- : u . .: .5 .. . V 1‘ act-on requires very high concentrations 6. terrocyahLQe (P9) tends to confirm the Chemiosmotic n LlCn that Phottrystcn 1: ;xioat1:n: : .3. v -... ‘ -~ OCCur -hsl.e of the th.lakoii w morgue. 'XANA, oar, GOULD [\‘(D “WC." A similar corrolat‘cv oetvecn nnoton "vizgcticn hr 1 ; i?‘“fi- \l -7 0 tion of L1.e -- hos been onsvrved W“Oh :,ho“ avert-Ju‘ J ‘ v; urn oxiiized by ”notosvstnn f1. ’n 2+0 “30*“ n” “"0 ~"‘-* “*‘~ *“ - s- . V - .- n .. : , .' - o." ‘ phosphorylatlcn, *9 WCllj 3:gn tn1: oc;fiuz rro u. “ro,Jr—lnogu¢nw~ such as nYdrOQULnOueS 1n: Dfi'tiuino 'HV'P1114; 29"mf in ‘30P”3 . ¢- ' . ~2 '* , - - .- .' - conserva,;cn at b-19 -. .n QJnZP«JL, w:iu;t-wn' _t 5:1: ‘ nwtai _ ‘. .. h . . . compleXcS of CI n:-:uv {0&3 du noo l.r0~,;v al-‘v- g -'o :r 1;“ ! -- '. o— 1 - n , . . .. . ' A . .. . .‘ , . tion ano ox;Ja.-ono of .wv. :uo;1¢.~ n «ILJr- fL. .. .n “‘2"“: . '- 4? ~ . as “I "' ChOQDhOPy_4C-uL av o-:9 -.. m, , a a , ‘ v 74"" x . , . ‘ . "N . _ , . _ .able 1. Phoopnor;;'t-3n L. -3- v-y A“ : :nc:-;n wt 23c Donor of 1-09tron: Lo FM a::3"':n 1’. (a ~ ‘ .'-n\ V‘ m *- Po ‘1 oonor ‘onc. ‘-- u.-.* ; 9’ I. ,_ , v. I V \\ ')I_.} — .a- _} , . I‘- - , ., Catecnol 4.9 . ‘- a.. v. ) 3 —. - ,~ ‘ r p-n;oroquonono v.3 pJ F.‘ v . ~ . r ‘ c-snLnCprcno; U.$ A“ ;. ’ ‘ q: ~ . ‘ 1.:n:L\|-nk‘ ..'. ; ‘C‘ 5" . ' . ‘ . ' . '1 ‘ '7 1 ”‘ Qin'JroxyoiphéuvL -.d (“ _.-« ‘ ,,‘ A _ CV t J.) vo‘: v Ferron\an*de ‘“ ’”' ” " ..J« ..A‘ .‘~ .-‘ ,. - . q , ‘ V 1 ~ r. ~ u Fe(d¢p;r-xv-h** 1.5 . H.‘. ‘, ' H V_ ‘. J r— r- I‘ v1n"3;.121~-,‘3** 3.1“ 1““;- _ .111 I : . ..2 ‘ r- ‘ - - .4n(g1-pgu -zufl)')** 0.:, .%n J. 2“ _ - *T?‘ — .v- — . ‘ ,x . ,‘ _- ,,: . ', -1 v _ “-92onL trlnhp.r, 4zfiaaz/.:.l ,H-, **Jc:onjarv P22?ZiP“R 33~02:3:~d .izh °nv .z-:a:i:” 3? LEHBS" vv\*£?l Cinnyoi': "Hm :‘~,..nnr a 'U v°— u- c m.,_ c«. . --’-~ —' U J C n1 (av ‘Ls ... AL‘L (.1 Cd n _v ..A. wit ': 1 o ..'.-.) s-’ .4 ' ;) v‘-- - .. _ . fl 9 . - ‘2 .. ' H. , _,, the time-coaroe of phobosnn‘int-‘n gt ;-te ; aKJ a: 9:2: :L, Lo.r at pH 6.5 and rH \ fl ..'. IZAHA, CRT COULD AND CO”? r l T V r’ ' Y HZO-fi'DMQ (Siteil) Ferrocy.~4-NFJ (Site!) nmomsATP/ml tJLn———hQ-f_—T——o.-l /pH 8.C.J pH 6. 1 5 O O 2 4 6 8 IO Time (sec) Figgre S. Phospharv as a ‘urctlon the time of illumiration. Clearly phosphorylation is linear with time and t ereforn musthave reached full efficiency within a small fraction of a atond Actu- ally, there is reaSOn to believe that nnosphorylatien starts Vezy much sooner than this. The Flash experiments of Bone? ari Mitt (37) suggest that phosphorylation occurs long b;fcre there is any appreciable difference between the concentrations \f nroton; inside and outside the thylakoid. Therefore, if the escape of protons from the inner aqueous phase is to drive phosphorylation, the prer'r:;nust *r! it( be under the impetus of a large, .tron- very e brane potential. 5 produce the same the oxidati of 'V t. produce the same m- then is there no when ferrocyanide are a membrane rather than preton accumula l ., A The second problem raised by f At iven by event: the chemiosmotic point of View. dr a good rate whether . At pH 6.5 or lower phosphorylation ( apace but phosphorylation driven by drOp in Site I phosphorylation at pH electron decrease in transport. unsnort-in dueedfmm- mirth“ .3“ ‘ Ann; .( ‘Abb potential 1.61. ITZAWA, ORT, Gnl'LD .-k\'D GOOD 657 the efficiency with which electron transport supports phosphory- lation. As figure 6 shows, the P/eD at Site - is very sen to'changes in pH whereas the P/92 ratio at Site II is quite in- different. V I I l T I I 1 0 I.o- o 0 0 3¢H20~Mv l Sites I+Il H2049“ P/ez o A C15*- 90 _ “‘Ferroc -. -——-—--—=\ w ’ /. \ : A HZO“. —;9-__’@/’ I ' DBNHB pH igure 6. Photophosphorylation efficiency as a function of the external pH. s The sensitivity of Site I phosphorylation to low pH seems to be independent of the chemical nature of the electron donor used: ferrocyanide (anion of a strong acid); DCIPHj (anion of a weak acid); DAD (a weak base). This naxes it extremely i mprobable that an identical uncoupling effect (due to the donors themselves) is re- sponsible for the low pH inhibition. It is also important to point out that these pH dependence curves are not likely to be the trivial consequences of any other abnormal aspects of the reaction conditions employed. The sum of the curves for Site I and Site I: phosphoryla- tion efficiencies, regardless of thc.electron acceptor: and electron donors used, is very close to the curve for the efficienc" of the normal Hill reaction which uses both Site I and Site II (see Consequently it seems safe to conclude tha the pH profile i and Site II,as measured by partial reazti approximate the true pH dependencies of t they are operating in concert in the ove The conclusion that Site II is resn phorylation below pH 6.5 is very difficul chemiosmotic model. Proton uptake associ 458 IZAWA, om, some mm mm) , 4 u . ~t. .: - . . ~ 1 u "x 7;, . ’ :+ .,"' ,, ' \ act- ns it very act-ve, witn a prohau_o “.:.A-tnuv L /u rit-o about 1.0 reflurdless of ph. aSee firure r). Lulwed the ef“::; of protor accumulation stems indeteni:'t of " un,a tie electvr' ‘ " ' ’4 0 .I '— , w ‘r 6— ' ‘ ‘ transport is tnrouvh either Site. .04 tne" r.n -. ,6 ..l. t~-om M4 ...~ 4»’. 4 |.O.uu—___-_---_------____-__ (Sifell) 01 Q) \ a + h H/e ‘3 .._-..----_---.O_9___------ m 0.5 / +\ I ’0 0C /—%. .Wh. . C \ P/ez FeN'OCy. —-vIV.V H /e (Site!) 6 7 8 pH Fiwure 7. A comparison of the R'V" y.. -',‘ n - ~ . :'—‘0‘ 13' and ALF syntncs s at 4-.-erent pi 3 question are very important. If we accent the contentitn thita cunu lated protons are uceJ to drive phosphorylation "e or: fnxed wi'n a chemiosmotic conundrum: Utilirat on of the a:cu~.l::~d 'rcton: “or phosphorylation seems to depani on how t - nrt;:r 1“ accun'7't2i. Clearly this cannot be if both Site I and ,;~u if ucritit tie grct3. '3 \‘u C) '1 "3C1UIOH‘D "3v 0) (D (D a orec*m U3 C) Q 0 k Mitochondr 32. TPCDSC, A. (l 33. Rxmberg, 3., Progress in Pho :1] \1‘) 4'\ r\ A (D N;L)r-‘ >——J . Yamqsnlta, T. an A., Ear” 7 “efe ’6) Biochim. ‘1, PHI... .1113. "i nd fiartin, 1. n3 Eutlnr, xawa, S. {197 \. p H °'fflC-, I‘., «‘r 7‘ So ". (l J.. (... . 106 ) . i J. an‘ Avr (1963) Pro tte‘ t A c r Neumann, J hence 3. 41 , 9.83'1'3 ICC rch Vol. I | ramer, N. . 2‘ w» .x :1, :Jo, Jo'-‘q akul, R., I: , 3204. d, J.M., Or Biophys. A Reimer, Reimer, Izawa, D.R. (V J n ..‘ ‘fi 0 H “or‘. rt, 956) Biol. 966) in Reg saglierello w) Ann. Rev . l P xxO \Jt—JOJF—Ictv $1 (‘0 +10 "J «1 W C; r.) Ho\o J < I 1 O \I‘J x"?- 5:11 o" n o. A 1 ‘x r a .‘\Cta L‘s, J87. I ‘r ’ . .‘ v- . k-4~9) 8-0:hln. :iooh a. 1 \ :1 e “: . ~ ~ '1'? :‘2 -3 ..1:. .hJSlO;. ~ , 1:5. '2 —~-.-:. r.‘: 46.) Pl ': :nys_cl. ~~, fl). - .. . :< "r Y .JF-l v':)~OLo if , 375- 'l w "v ‘ n ‘v . .\ er, a. -0« , e. ”QCJ? orscn "‘ -’" ' o- ‘: ' , .23; et.ers 20, 1 l {1 ""1 r) n, , .~ . *’ oi A. -f')l‘\ {4. xl‘)-t 1.” 0'1 Cfl.(‘.-,_)“l "I . u- .. . ’ - ‘-'- : :’¢QC‘li'1n :‘LC‘:.'h:I'I‘-o PU: . )- _1'4z',l’3-) " r‘.\'. V.. r- r ‘4..- 1.. 1."! “')v.1..:4'., J. L ‘ j» v f‘ I “'?/'\ "I up nova, o. \“..) H. ..lanzen- -ochem. Bloony.. ‘0». Commlr. r) : ‘ g "A .ldnt .h'n-0-. J?, _ -. “v ' 4 .. 1:. _. . " ’7 (2‘ 5-0: -.. -.oph.:. .cta 3.5,520. (’J .1 « . -m. --t 1 Fist)" ' ,l ‘ t -.~"4 _l‘ v ErO‘xhevcn q- .7) n , ' I‘ll: o - 16 o eds) 813 l 165 'hosphoryla: LLUP. I.) o B: ,1 I Tetab ion. -'I'«'“ lu‘-JO 0 ;‘ylt' .... \"x-mn k ..AL: . .. .» J} ’ . olic .l ‘I 4" ._€-, d a side-reactlon _‘IHJJ- ‘-‘-“ r‘fl ~\ :1“) JL O‘J-\.&l‘fiy action and . .er space in F . Jyfi.<~ IMP} , . . ‘_.\¢J‘Al .J‘)4,:/ ’ . ;nner spa: -\ ‘ 1 l o ‘ _‘Jmfin'ltl'dn ~.: ‘ ¢.. . 'zJ!!-C.'l .':- CE": "I ”\ ‘ -‘ 0 'r‘. 1. ~ -.J-. 1.9, -49 ‘ " "”‘S ...'1 N01: V P!- “'5 , --— I. I ..‘.y \ V‘" "c K-“ f... (w-fi'“ r aka 4’ ‘i' f‘ . I " IC«.‘.., I054. F ’ f‘r - nCva Ru'l’ a. ’724 .."'\. ..ep, (..‘... 3... ,CE. 5, , .LJ,‘. —.—-~ «‘1 n " A-t.;. 5)., .Lc. scesses ir Tr, ,. 0 L61 6 ‘3’ _‘ ..o . 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