.4 I f“ m d 'v-' Okfiw “.- n“, w. h“;....; L125 KARY Michzgm t in: “a. ’ *3}, .44 "'~""""'-"v-ma-“ni 8 This is to certify that the thesis entitled THE KINETICS OF ELECTRON TRANSFER REACTIONS IN CHLOROPLAST PHOTOSYSTEM II presented by Christine Thompson Yerkes has been accepted towards fulfillment of the requirements for M. S. Jegee in Chemistry mé/mp/ Major professor Date 7/4 #9 / 0-7639 i_Lw W: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return toremo remove charge from circulation records THE KINETICS OF ELECTRON TRANSFER REACTIONS IN CHLOROPLAST PHOTOSYSTEM II By Christine Thompson Yerkes A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 1979 ABSTRACT THE KINETICS OF ELECTRON TRANSFER REACTIONS IN CHLOROPLAST PHOTOSYSTEM II By Christine Thompson Yerkes The rate of photosynthetic oxygen evolution, measured polaro— graphically under conditions of continuous illumination, is examined as a function of pH in the presence of a variety of uncoupling agents. A pH optimum is observed at about pH = 7.0 for all cases. A model has been developed and tested to explain the uncoupler mode of action and the complicating role of the membrane. To define the origin of the observed pH dependence of oxygen evolution, analogous experiments have been carried out using the EPR detectable species, signal IIf, as a probe for the electron transfer kinetics on the water side of Photosystem II, the site of oxygen evolution. A model has been developed to explain the pH dependence of signal IIf decay kinetics in the presence of ascorbate. The kinetic model shows good agreement with the experimental data if either a pH dependent proton gradient across the membrane or a negative interior surface charge is postulated. ACKNOWLEDGEMENTS I would like to thank Martin Rabb for his technical assistance, the staff of the electronics shop for their patience and especially Professor Jerry Babcock for his guidance in this project. Its existence attests to the fact that his enthusiasm and scientific curiosity finally proved contagious. ii TABLE OF CONTENTS List of Tables . . . . . . . . . . . . . . . . . . . . . . . . v List of Figures . . . . . . . . . . . . . . . . . . . . . . . vi List of Abbreviations . . . . . . . . . . . . . . . . . . . .viii INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . 1 I. The pH Dependence of Photosynthetic Oxygen Evolution . . . 1A A. Introduction . . . . . . . . . . . . . . . . . . . . 1A B. Materials and Methods . . . . . . . . . . . . . . . . 1A 1. Light Source and Optical Arrangement . . . . . . 1H 2. Reaction Chamber and Oxygen Probe . . . . . . . 15 3. Detection and Recording Circuits . . . . . . . . 16 A. Spinach and Chloroplast Suspensions . . . . . . 16 5. Experimental Protocol . . . . . . . . . . . . . 16 C. Results . . . . . . . . . . . . . . . . . . . . . . 21 1. Polarogram . . . . . . . . . . . . . . . . . . . 21 2. Calibration of the Electrode . . . . . . . . . . 2H 3. Saturation Curve . . . . . . . . . . . . . . . . 2A A. The pH Dependence of Oxygen Evolution . . . . . 2h D. Discussion . . . . . . . . . . . . . . . . . . . . . 31 II. The pH Dependence of Signal II no f . A. Introduction . . . . . . . . . . . . . . . . . . . . “0 iii B. Materials and Methods . . . . 1. 2. 3. Kinetic EPR Instrumentation Spinach and Chloroplast Suspensions Experimental Protocol . . C. Results . . 1. 2. Signal I and Signal II . . Signal IIf . D. Discussion . . . . . . . . . 1. 2. BIBLIOGRAPHY Endogenous Electron Donors . Exogenous Electron Donors iv In 1:2 as us as us 119 61 61 65 78 Table 1. LIST OF TABLES A breakdown of the bi-phasic decay of signal IIf in the absence of exogenous donors; k8 = slow decay rate, k1. = fast decay rate, AS = percentage of total signal following slow kinetics, Ar = percentage of the total signal following fast kinetics . . . . . . . . . . . . 6h Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 10. 11. 12. 13. 1H. 15. LIST OF FIGURES The basic elements of the Z-scheme, two light reaction hypothesis for green plant photo- synthesis . . . . . . . . . . . . . A representation of the thylakoid membrane showing component asymmetry . . . . . . Oxygen evolution detection and recording circuit Schematic representation of the oxygen evolution detection system . . . . . . . . . . . . . . Polarogram of air saturated buffer . . . . . Calibration curve for the oxygen electrode Saturation curve Relative rates of oxygen evolutfion vs. pH for (e) a system uncoupled with M NHuCl, (A) a system uncoupled with 103 M _gH _NH2, and (I) a system uncoupled with 10 R A plot of ApH vs. pH for amines with different pKa values . . . . . . . . . . . . . . . . . A plot of amine (CHBNHZ) concentration vs. calculated ApH . . . . . . . . . . Circuit diagram for diode opto-isolator . Block diagram of kinetic EPR instrumentation EPR spectra of TRIS washed (pH = 6.5) chloro- plasts during (a) and after (b) illumination. . The effect of exogenous electron donors on EPR signal 11f decay kinetics . . . . . . . . . A semi-log plot fo the natural log of signal IIf concentration vs. time at pH : 7.5 in the absence of exogenous electron donors . vi gramidicin . 20 23 26 28 30 35 37 A” A7 51 53 55 Figure 16. A semi-log plot of the natural log of signal II concentration vs. time at pH = 6.5 with an added ascorbate concentration of 2 x 10 Figure 17. A plot of the observed decay rate of signal IIf (ko ) vs. concentration of ascorbate at two BIfferent pH values . . . . . . . . Figure 18. A plot of signal II decay half-times vs. pH for three experimental systems . . . . . . . Figure 19. A plot to the con entration of neutral ascorbate (solution 2 x 10 'M on ascorbic acid) vs. pH (0) and a plot of kobs vs. pH (0) . . . . . . . Figure 20. A plot of koalc/kobs for two different chloro- ons . plast suspens . . . . . . . . . . . . . Figure 21. A plot of ApH vs. external pH calculated from the obsgrved decay kinetics of signal IIf with 2 x 10' [M ascorbic acid as an exogenous electron donor. . . . . . . . . . . . . vii _ADP ATP Asc Chl Cyt EDTA EPR Fd HAsc HEPES HQ MES NADP NADPH Pi PCy PQ PS I PS II TRICINE TRIS X ABBREVIATIONS Adenosine diphosphate Adenosine triphosphate Ascorbate Chlorophyll Cytochrome Ethylenediaminetetraacetic acid Electromotive force Electron Paramagnetic Resonance Ferredoxin Ascorbic acid N-2-Hydroxyethy1piperazine-N'-2-ethanosulfonic acid Hydroquinone 2(N-morphalino)ethanesulfonic acid Nicotinamide adenine dinucleotide phosphate Nictoinamide adenine dinucleotide phosphate, reduced form Inorganic phosphorous Plastocyanin Plastoquinone Photosystem I Photosystem II N-tris(hydroxymethyl)methylglycine Tris(hydroxymethyl)aminomethane Magnetic susceptibility viii INTRODUCTION Although many advances have been made in understanding the photo- synthetic process in bacteria and higher organisms, the photosynthetic evolution of oxygen remains a major process whose mechanism has not been explained. The role of oxygen evolution relative to the other light driven reactions in photosynthesis is described in Figure 1, a schematic representation of the overall process of higher plant photosynthesis. This model, often referred to as the Z scheme, can be conveniently divided into two photochemical systems: Photosystem I (PS I) associated with the reduction of NADP and Photosystem II (PS II) associated with the oxidation of water. The light harvesting components, referred to as antenna pigments, are capable of trans- ferring energy via both exciton interaction and Forster transfer (Sauer, 1978) to specialized molecules called reaction centers. These two forms of chlorophyll, antenna pigments and reaction centers, exist in a ratio of about A00 to 1 (Emerson and Arnold, 1932). Light energy absorbed by the light harvesting system of PS II is trapped at a reaction center called P680. The name of this component is derived from its presumed absorption maximum at 680 nm. Light induced charge separation at this reaction center leads to the reduction Figure 1. The basic elements of the Z-scheme, two light reaction hypothesis for green plant photosynthesis. Vertical arrows refer to changes in relative reducing potential. Abbreviations: Z, the primary donor of PS II; P680, the PS II reaction center: Q, the primary acceptor of PS II; PQ, Plastoquinone; cyt. f., cytochrome f; PCy, Plastocyanin; P700, the PS I reaction center; X, the primary acceptor of PS I; Fd, ferredoxin; Fd-NADP red., ferredoxin-NADP reductase. ”Q4 00 E°(V) +04 +08 F §>0 / Ll P680 21420) 2 02+ 4H+ PO ADP ctt PC ATP Y \ >>< / Tl Ffi NAIDFD ed NADF3 6 A M hl’], P700 of a primary electron acceptor, Q, (E ~ 0.0 V) and the oxidation of P680 to P680+. The species Q is thought to be a plastoquinone molecule. The subsequent reduction of P680+ leads to the production of a strong oxidant, 2+, (E ~ +0.8 V) which is as yet unidentified chemically, capable of oxidizing water to molecular oxygen. Similarly, light energy absorbed by the antenna pigments of PS I is trapped at a reaction center which contains another photo- oxidizable form of chlorophyll, P700. Again, its name describes its absorption maximum near 700 nm. Charge separation at the PS I reaction center leads to the oxidation of P700 (E ~ +0.“ V) and the reduction of a primary electron acceptor, X, (E ~ -0.6 V). The reduced form of Q in PS II and the oxidized form of P700 in PS I are re-oxidized and re-reduced as a result of electron transfer along the electron transport chain connecting the two photosystems. The net result of the photoexcitation of these two photosystems is the transfer of electrons from water to the species X which then mediates the reduction of NADP. This links the oxidation of water to the formation of NADPH, a species used to fuel the enzyme cycles associated with the assimilation of C02 in the dark, exothermic reactions of photosynthesis. An important property of the photosynthetic light reactions that is not evident from the Z scheme is its vectorial nature. The photosynthetic organelle of higher plant photosynthesis is the chloro- plast. The pigments and enzymes required for the light dependent reactions are localized on the membranes of flattened sacks within the chlorOplast called thylakoids. The thylakoid membrane, as part of a closed vesicle, separates two chemically different environments, the outside or matrix side from the inside. The distribution of the photosynthetic components within the membrane is asymmetric, giving rise to a directionality in the electron flow. These asymmetric electron transport reactions result in both charge and proton gradients across the photosynthetic membrane which ultimately provide the free energy required for ATP synthesis (Mitchell, 1966; Neumann and Jagen- dorf, 196“). Figure 2 is a representation of the thylakoid membrane which incorporates membrane asymmetry in a schematic manner. The exact placement of the components is an area of very active current research, making the arrangement shown in Figure 2 tentative. None- theless, it is consistent with most of the experimental results reported to date. Those factors especially pertinent to the under- standing of PS II are the presence of the plastoquinone pool and the location of P680 and 2 toward the inside of the thylakoid. Plastoquinone serves as a secondary electron acceptor in the sequence of reactions following the photooxidation of P680. Each molecule of plastoquinone can accept two electrons as well as two protons from the chloroplast matrix. The lipid soluble reduced plastoquinone may then diffuse through the membrane, deliver two electrons to the next acceptor, currently though to be cytochrome f, along the electron transport chain joining the two photosystems and finally release two protons to the interior of the thylakoid. This results in the net influx of two protons for each molecule of plastoquinone reduced and subsequently re-oxidized. Another source contributing to the accumulation of protons within the thylakoid is the oxidation of water. For every mole of molecular oxygen produced, four moles of Figure 2. A representation of the thylakoid membrane showing component asymmetry. Abbreviations: chl a, antenna pigments; Z, the primary donor of PS II; P680, the PS II reaction center; Q, the primary acceptor of PS II; PQ, the transmembrane, plastoquinone pool; cyt. f., cytochrome f; PCy, plastocyanin; P700, the PS I reaction center; X, the primary accepter of PS I; Fd,ferredoxin; Fd-NADP Red., ferredoxin-NADP reductase. thylakoid membrane outside H. @ NADP inside p 13 0- NADPH m 4? am. @ hv protons are released to the inside of the thylakoid. Through these two proton translocation sites, as well as others associated with PS I, a proton concentration gradient is established and maintained across the membrane. 0f the two light harvesting systems, the understanding of PS II has evolved more slowly than that of PS I. Indeed, as recently as 1966, the photoreaction of PS II was described as the "inner sanctum" of photosynthesis (Kok and Cheniae, 1966). Fortunately, since then advances have been made and although knowledge of the electron transport pathways and the photochemical reaction remains incomplete, the following model of the PS II reaction sequence has evolved: + a 1) chla + hv chla Light absorption by antenna chlorophyll. 2) chla. + P680 Q -+ chla + P680‘Q Transfer of excitation energy from the antenna chlorophyll to the reaction center. a - 3) P680 Q + P680+Q Charge separation at the reaction center. 11) z P680+Q-R + z+P680 Q-R Electron transfer from donor 2 to P680. + - + - 5) Z P680 0 R +' Z P680 0 R Electron transfer to secondary acceptor R linked to plastoquinone pool. + 6) Sn + Z -+ Sn+1 + 2 Charge accumulation, n=0, 1, 2, 3. + 7) 2H20 + SN +- “H + 02 + SO Oxygen evolution. Reactions 1) and 2) occur in picoseconds, 3) in less than 20 nano- seconds, A) in less than a microsecond and 5) with a half-time of about 600 microseconds. A brief description of reactions 6) and 7) follows. Measurements by Joliot gt 2l° (1969, 1971) and Kok gt El: (1970) of oxygen evolution produced by short (duration less than 10 us) saturating flashes show that the amount of oxygen produced by chloro- plasts that have been in darkness for 15 min is different for each flash. Little or no oxygen is produced by the first two flashes and a maximum is observed after the third flash. A continued series of flashes gives a damped oscillation with a period of four when oxygen yield is plotted against flash number. These results have been interpreted by Kok gt El- (1970) in the following scheme: hV ' hv * hV . hV ' 30+ 30+ 31+ 31+ 32+ 32 + 33+ 33 + S“ l + 02 + NH 2H20 a a The steps Sn 2» Sn represent phototransitions and the steps Sn + Sn+1 are the subsequent dark electron transfer steps. The kinetics of these charge transfer reactions are slow compared to the 10 us flash duration, hence the ability of a single flash to promote a 10 single electron transfer. Oxygen evolution occurs after storage of four oxidizing equivalents. The maximum yield of oxygen after the third flash indicates that most of the reaction centers are in the S1 state after dark adaptation. It has been postulated that both the SO and S1 states are stable in the dark and exist in a ratio of 1 to 3. The damping of the oscillatory pattern arises from "misses", a percentage of failures to produce charge separation at each reaction center (roughly 10% per flash). Little is known about the oxygen evolving step except that it has been shown that membrane bound manganese is involved (Cheniae and Martin, 1966; Blankenship and Sauer, 1974). Presumably, the charge accumulating reactions correspond to oxidation of the manganese species, although no firm experimental evidence exists to support this supposition. The kinetic data and the model described above are based on data gathered by four techniques, oxygen evolution in flashing light and fluorescence, luminescence and absorption spectroscopy. Fluores- cence emission with a quantum yield of about 0.03 is observed in functioning chloroplasts. Duysen and Sweers (1963) have demonstrated that the reduction and oxidation of Q, the primary electron acceptor of PS II, causes a corresponding increase or decrease in the fluores- cence yield of the antenna chlorophyll. Oxidized Q is associated with low chlorophyll fluorescence, an observation which has led to the assignment of the term "quencher" to the species 0. An additional hypothesis, proposed by Butler 23 El: (1973) on the basis of experi- ments at 77 K, is that not only 0 but also P680+ quenches fluores- cence. This postulate explains the low fluorescence yield observed 11 for both the P680 Q and 9680*0' states. Although the range of validity of this working hypothesis has not been rigorously established, it is a useful tool in the study of P680+ reduction kinetics. Immediately after a flash, when the system is in the P680+Q- state, the fluores- cence yield is low, due to the quenching of P680+. The fluorescence yield will subsequently rise due to reaction A) which leads to Z+P680 Q'R, a state with no quenchers. The kinetics of the rise in fluores- cence are interpreted as the rate of reduction of P680+. Another method of studying the kinetics of P680 is through luminescence or delayed light emission, which gives a typical quantum A 5 yield of 10- to 10- . Lavorel (1975) has hypothesized that the delayed light arises from the recombination reaction: P680+Q' + p6ao'c + P680 Q + hv The species P680+ is the lowest excited singlet state of P680. Dark regeneration of this state leads to the delayed emission of light. Luminescence intensity is then proportional to the concentration of P680+Q-. Since 0' is re-oxidized much more slowly than P680+ is reduced, the decay rate of the luminescence is proporational to the reduction rate of P680+. Another technique used in the study of P680 kinetics is absorp- tion spectroscopy. Until recently, no absorption changes attributable to the reaction center of PS II could be observed under physiological conditions. At low temperatures or in the presence of detergents, the reduction of photooxidized P680 and the re-oxidation of Q- can be slowed down. Floyd gt_§l. (1971) observed a bleaching at 680 nm 12 upon illumination of chloroplasts with short laser pulses at 77 K. This absorbance change decays with a half-time of ”-5 ms upon darkening. Von Gorkom gt §l° (1974) observed the photooxidation of P680 in chloro- plasts treated with the detergent deoxycholate and in the presence of ferricyanide, have found that the reduction of P680+ is slowed to such an extent that an appreciable amount of P680+ can actually be accumulated under conditions of continuous illumination. Recent work done by Van Best and Mathis (1978) allows the study of the reduction kinetics of P680+ at physiological temperatures. Their development of a flash absorption photometer with nanosecond time resolution has made this possible. They have found a fast (30 ns) decay time for P680+, reflecting its reduction by the secondary electron donor. Of all of the techniques mentioned, this is the only one which measures a kinetic rate on the oxygen evolution side of P680. It is the intent of this study to investigate an additional means of collecting data directly from the electron transport system used to reduce P680+. The recent observation and characterization of an EPR detectable species (Babcock and Sauer, 1975; Blankenship 33 al., 1975) provides an excellent tool in studying these complex kinetics. The EPR active species appears to function as an intermediate in the electron trans- port process between P680+ and the Sn states. In active, oxygen evolving chloroplasts, this signal is observed within 20 us of an actinic flash. Its decay, under steady state illumination conditions of roughly one flash per second, has a half-time of about 600 us. However, upon inhibition of oxygen evolution, brought about by TRIS 13 washing or exposure to heat, the decay rate falls dramatically to give a half-time of about 1 s. Introduction of artificial electron donors (phenylenediamine, hydroquinone, benzidine) to the inhibited chloroplast suspensions accelerates the decay process. The rapidly decaying signal from uninhibited chloroplasts has been named signal IIvf (very fast) while the signal associated with the inhibited chloro- plasts has been named signal IIf (fast). It is the eventual intent of this research project to determine if both signals arise from one species, Z, the variation in decay kinetics reflecting the nature of the final electron donor. That is, if Z+ is reduced by the water oxidizing enzyme complex (Sn states) and water, then signal IIvf if observed. If, however, water oxidation is inhibited, then 2+ is reduced by endogenous electron donors and signal IIf is observed. It if can be shown that these two signals arise from a common species which exists as an intermediate in the electron transport chain between P680 and the Sn complex, an experimental observable will be available for the first time for the study of the complex reactions which lead to the photosynthetic evolution of oxygen. In the initial experiments designed to probe this system, we have carried out a study of the effect of pH variation on the overall process of oxygen evolution. Parameters which complicate this process, particularly the membrane pH gradient, have been explored. Analogous experiments which use the EPR detectable species as a probe for electron transfer kinetics on the water side of PS II have been carried out. I. The pH Dependence of Photosynthetic Oxygen Evolution A. Introduction The study of oxygen evolution as a function of pH is especially important since one effect of the light reactions of photosynthesis is the establishment and maintenance of a proton gradient across the thylakoid membrane. Variation of the pH of the suspension medium also offers a convenient and systematic way to perturb the system. Membrane processes, however, complicate the system to some extent since protons are released to the interior of the thylakoid during the water oxidation reactions. Simultaneously, protons are carried through the membrane by the plastoquinone pool. The overall result is a pH gradient across the membrane. In normally functioning chloro- plasts, phosphorylation reactions, ADP + Pi + ATP, are coupled to the efflux of protons from the thylakoid interior and dissipate the pH gradient (Mitchell, 1966). During chloroplast isolation, however, the soluble phosphorylating factors are removed. To prevent excessive acidification of the thylakoid interior, uncoupling agents have been used to replace the function of phosphorylation (Good, 1960; Hanstein, 1976). Because PS II is effectively isolated from PS I by this treat- ment, the addition of an exogenous electron acceptor is also necessary. In the experiments described below, we have investigated oxygen evolu- tion as a function of pH and uncoupler mode of action. B. Materials and Methods 1. Light Source and Optical Arrangement The continous light source for this series of experiments is a General Electric 200 watt projection lamp. The intensity of the light reaching the sample is 5.8 x 105 erg cm"1 3'1 as determined 1“ 15 by a calibrated PIN 8 photocell. The light is passed through a heat absorbing filter (2 in x 2 in, Corning) and focused on the reaction chamber by a pair of planoconvex lenses. A set of neutral density filters (Balzer) allows controlled attenuation of the light incident on the reaction chamber. 2. Reaction Chamber and Oxygen Probe The reaction chamber is a lucite cylinder of .75 cm radius and 6 cm height encased in a lucite cube (7.5 cm on a side) through which is flowed controlled temperature water from a model 2095 bath circulator (Forma Scientific). A lucite plug seals the reaction chamber after addition of the reactants. The tip of the oxygen probe lies flush with the side of the reaction chamber and perpendicular to the path of the light to prevent direct illumination of the electrodes. The probe, model YSIS331, Yellow Springs Instru- ment Company, consists of a platinum cathode and a silver anode which are bathed by a KCl solution held in place by a Teflon membrane bound to the probe tip. When a polarizing voltage is applied across the probe, the oxygen in the vicinity of the cathode is reduced according to the reaction: + 2H 0 + ue" + 1103' O2 2 The corresponding anode reaction is: llAg + MCl- + UAgCl + Lle- Current flows in direct stoichiometric relation to the rate of oxygen reduction. As the oxygen is depleted in the region adjacent to the cathode, oxygen in solution diffuses through the Teflon membrane 16 at a rate proportional to its concentration. To insure a uniform oxygen concentration throughout the reaction chamber, constant stirring is provided by a synchronous motor operating a small magnetic stir bar in the sample chamber. 3. Detection and Recording Circuits The circuit used to provide a bias to the electrode and to detect oxygen evolution is shown in Figure 3. The main com- ponent is a current to voltage transducing operational amplifier (Signetics-pA7A1). The output voltage is recorded as a function of time on a Sargent recorder model TR S-72190. A schematic repre- sentation of the oxygen evolution detection system is shown in Figure A. A. Spinach and Chloroplast Suspensions The spinach used in these experiments is laboratory grown in a growth chamber (Environmental Growth Chambers). The plants are subjected to day and night cycles of 10 hours of light followed by 1” hours of darkness. The spinach is grown in an inert vermiculite base, the nutritional requirements being met by watering them with Modified Hoagland's Nutrient Medium (Hoagland and Brayer, 1936). The temperature in the growth chamber is maintained at 16°C. A simple and rapid process for chloroplast preparation is used (Babcock and Sauer, 1973). The chlorophyll concentration is determined by the method of Sun and Sauer (1971). 5. Experimental Protocol In the dark, 3.5 ml of reaction solution is placed in the reaction chamber. This solution is 20 mM in NaCl, 5 mM in MgClz, 1 mM in K Fe(CN)6, 1 mM in KuFe(CN)6, 50 mM in buffer and 10 m! in 3 17 .uwsocflo wcflucoomc new soapomumu cowusao>m comzxo .m masmflm 18 6334 4—.“ a (I So>1 n 3.. n Flv 5:1 I a. .Em o m 3: m _ l l a. :R >n_+ acme o _ 4 av: QXMOM 4Sims. .0. $00— 19 .smpmhm cowuooumu coflusao>m cmwmxo on» go coflumucmmmeamg ofimemsom .= mesmflm 20 Ikdm c mks: wmakdmwnZMF owJJothOU ado Rpm 1.:3 >._..>eso coaumnzumm .u omsmwm 28 CE: 2:265 2.23:. 29.. OO. 00 on Oh 00 00 0? On ON 0. A m _ _ _ _ _ _ _ _ (suun GAHDIGH) U0!ll’1|Ol\3 uablixo To 3:03 29 Figure 8. Relative rates of oxygen evolution vs. pH for (o) a system uncoupled with 10'3 M NHuCl, (a) a system uncoupled with 10-3 ‘! CH3NH2, and (a) a system uncoupled with 10-6 M gramidicin. [1. mole 02 30 mg chlorophyll h J 1 1 1 ‘ ‘ CH3NH2 0= NH3 '8 Gromicidin l 5.5 so 6.5 7.0 7,5 8.0 8.5 31 dramatically. This loss of oxygen evolution can be explained as the inactivation at high pH values of the S and S states of PS 2 3 II (Briantais 23 al., 1977). D. Discussion During photosynthetic oxygen evolution, a proton gradient is established across the thylakoid membrane by the translocation of protons from the matrix to the thylakoid interior and by the oxidation of water. It is possible to approximate the extent of this proton influx during the course of one experiment (30 s) from the measured rate of oxygen evolution. For the maximum rate observed, 1.02 x 10-8 moles of oxygen evolved in 30 3, about 1.6“ x 10‘"7 moles of protons are released to the insides of the thylakoid membranes. An approximate volume for the total inner thylakoid space is 0.5 ul/ml suspension or about 1.75 pl under our experimental conditions (Siefermann-Harms, 1978). Using these values, we estimate that an internal pH of 1 would result from proton release. Before such a physiologically damaging pH could develop, the rate of oxygen evolution would fall. In order to allow oxygen evolution to continue at a measurable rate in isolated chloroplasts, uncoupling agents have been added to replace phosphorylation as the counter reaction to the proton influx. One class of uncoupling agents, which has been found to be effective empirically, is amines. The neutral form of the amine is postulated to diffuse through the membrane to the interior where it may be protonated and act as an internal buffer (Crofts, 1967). A model is developed here to determine the internal buffering capacity 32 of various amines as functions of their pKa, their concentration and of the pH of the suspension medium. Our model depends on three assumptions: 1) Before illumination and the initiation of oxygen evolution, the amine concentration equilibrates across the membrane: a I [RH]i = [RH]o RH = neutral and protonated amine species. 9 a before illumination 1 = inside thylakoid. o : outside thylakoid. 2) During oxygen evolution, the instantaneous concentration of the neutral amine on the inside and outside of the membrane is the same: [R10 = [R11 R = neutral amine. 3) For every mole of protons released to the interior of the thylakoid, one mole of neutral amine will be taken into the thylakoid from the suspension medium: ' + F + + vlfinlo - vha]o + VIRH lo - Hp - v[11]o + vlnh 10 [an] - [31' v[nn*]' 3+ - v[R] . v[RH+] V 1 ' V 1 * 1 * p ' 1 1 V = volume of the suspension medium. v = volume of the inner thylakoid space. RH+ = protonated amine. H+ = the number of moles of protons that have been released to 33 the thylakoid interior as a result of photosynthetic reactions at the time the calculations are made. Using these equations along with the pKa and concentration of the amine and the pH of the suspension medium, one may calculate the internal pH as a function of the external pH. The difference between these two values is known in the literature as ApH. Figure 9 is a plot of calculated ApH vs. external pH for ammonium chloride (pKa = 9.2u9), methylamine (pKa = 10.569) and a hypothetical amine with a pKa of 7.0. If the external pH exceeds the pKa of the amine, the ApH deviates strongly from unity and the internal pH is no longer linearly related to the external pH. However, both ammonium chloride and methylamine maintain a ApH close to unity over the experimental pH range (5.5 - 8.5). These observations, based on the model developed here, explain the experimental observation that only amines with high pKa values are effective uncoupling agents (Schuldiner §t_al., 1971). It also explains why common buffers such as phosphate, HEPES, TRICINE and TRIS, with pKa values in the range 7.0 - 8.0, do not interfere with the phosphorylation process. Figure 10 is a plot of amine concentration vs. ApH. As the amine concentra- tion increases, pH approaches zero and the internal pH asymptotically approaches the external pH. Qualitatively, these calculated data agree quite well with experimental values obtained in a study in which no theoretical model was proposed (Rottenberg and Grunwald, 1972). The ammonium ion concentration used in our experiments is 10'2 M. A more concentrated solution, which would lower the ApH, was not used as it has been reported that high ammonium ion concentra- tions inhibit oxygen evolution (Delrieu, 1976). 311 Figure 9. A plot of ApH vs. pH for amines with different pKa values. 3.0 *— 25 — ApH ]l_ 11 0.0 35 °= Amine with pKo =7.0 x= Ammonia, pKo = 9.249 . = Methylcmine,pKo =lO.657 4 w 5.0 6.0 7.0 8.0 9.0 36 Figure 10. A plot of amine (CH3NH2) concentration vs. calculated ApH. 0.0 — 0.5 — 1.0 ‘- l5— 2.0 — 2.5 '- 3.0 — IXIO'4 M 37 l IO'3M I I l 10'2 M 10" M IO°M [C HsNHzl 38 The experimental results (Figure 8) show a definite relationship between observed rates of oxygen evolution and the pH of the suspension medium. Through our model we have determined that the efficiency of the uncoupling amine is relatively independent of the pH of the suspension medium when the pKa of the amine is sufficiently high (Figure 9). If this were not the case, however, the observed pH dependence of oxygen evolution might reflect an artifactual pH depen- dence of this system, perhaps in the rate of amine incorporation into the thylakoid. At low pH values, the concentration of the neutral amine in the suspension medium would be correspondingly low which could lead to a lower rate of transport across the membrane, a less efficient internal buffering capacity and eventually to a lower rate of oxygen evolution. To test this possibility, the experiments have been repeated using an uncoupling agent that works by an entirely different mechanism. The agent chosen is the antibiotic, gramicidin, which has been shown to uncouple phosphorylation by increasing the permeability of the membrane toward cations (Junge and Witt, 1968; Shavit 22 al., 1968). The photosynthetically induced proton gradient is dissipated by the outward diffusion of protons through the hydrophilic pores that result upon the incorporation of gramicidin into the membrane. Oxygen evolu- tion experiments using gramicidin as the uncoupling agent show the same pH dependence as those using amines (Figure 8). This leads to the conclusion that the incorporation of the amine is not the rate determining, pH dependent step in amine uncoupled oxygen evolu- tion. 39 The other component which could lead to an artifactual pH depen- dence is the exogenous electron acceptor, ferricyanide. Experiments by Itoh (1978) have shown that ferricyanide is photoreduced by a component of the electron transport chain linking the two photosystems. Itoh found that low pH values lead to higher rates of ferricyanide reduction. Based on this observation, he suggested that the ferri- cyanide reduction site is buried within the membrane and that negative charges at the membrane surface impede the diffusion of ferricyanide to the site. At low pH values, the surface charges are shielded, allowing easier access to the site. This pattern of decreasing rates A of ferricyanide reduction with increasing pH is opposite to the trend observed in the oxygen evolution experiments where the reaction rate increases with increasing pH. Thus, ferricyanide reduction does not appear to be the pH dependent, rate limiting step of oxygen evolution in this system. In these experiments, we have measured the pH dependence of oxygen evolution and obtained data in agreement with those of others (Holt and French, 19u8; Renger 92 al., 1977). We have developed and tested a model to explain the uncoupler mode of action and to define more clearly the complicating role of the membrane. Using the model and experimental results, we have shown that the observed pH dependence is a true property of oxygen evolution and not merely an artifact of the system studied. It is, however, a macroscopic property. The following experiments are an attempt to further define its origin. II. The pH Dependence of Signal IIf A. Introduction As described in more detail in the Introduction, this signal arises from a component on the water oxidizing side of P680. Its position in the electron transport sequence is shown in the scheme: ZHZO P680—z—Sn C + O +1111 2 where Z represents an unknown number of electron transport inter- mediates, one of which gives rise to an EPR signal. After photo- oxidation of P680, the Z species reduce P680+. During this sequence, one intermediate becomes EPR detectable upon oxidation; its subsequent reduction can be observed as the decay of the EPR signal. In oxygen evolving chloroplasts, the case illustrated above, the final reducing agent is water. Under these conditions, the EPR signal observed is called signal IIv It has a rise time of less than 20 us and f. a decay half-time of about 600 us. If, however, oxygen evolution is inhibited, the reaction sequence changes: inhibition 2H20 P680— 2 _“__s n endogenous or exogenous + electron donors The physiological electron transport pathway can be blocked by inhi- bitory techniques, among them TRIS washing, heating, aging or the addition of denaturants. These treatments release membrane bound manganese and result in the loss of oxygen evolution. Under these A0 N1 conditions, the ultimate P680+ reducing agent is no longer water, but endogenous or exogenous electron donors. Upon inhibition of oxygen evolution, the decay kinetics of the EPR signal are slowed considerably, the decay half-time changing to about 1 s. The EPR signal exhibiting these decay kinetics is called signal II Addition f. of exogenous electron donors decreases the decay half-time. B. Materials and Methods 1. Kinetic EPR Instrumentation a. EPR Spectrometer, Signal Averager and Timing Circuits All experiments are performed with a Varian model E-u spectro- meter using a transverse magnetic 110 mode cavity (Varian E238). The sample cell is a Scanlon flow EPR flat cell (S-81A). Because of the relatively low concentration of the paramagnetic species obtainable in the chloroplast suspensions, the time course for the transient rise and decay of the EPR detectable species must be enhanced through time averaging. Data are collected and stored with a Nicolet model 107A signal averager. A plug in model SD-72/2A analog to digital converter and model SW-71A time base are used in the 107“ main frame. A TTL logic-based timing circuit has been designed and built by the Michigan State University Chemistry Department electronics shop which allows us to trigger up to six different instruments at intervals continuously variable from 1 us to 100 Us. b. Laser The laser head, containing the main discharge capacitor, flash lamp, pump and dye cell, is a model DL-1100 from Phase-R Corporation, "2 New Durham, New Hampshire. It is rated at 250 mJ output light energy with rhodamine 60 (A m x = 590 nm) with a pulse duration of #50 ns. a The 25 KV power supply has been built by the electronics shop. The triggering system, based on a schematic supplied by Phase-R, was further developed in our laboratory. The first experiments using the laser light source revealed a large electrical transient, generated by the triggering of the laser, which obscured the EPR signal. The following shielding scheme evolved to separate the laser and EPR systems. The laser is in a Faraday cage with r.f. shielding (Instrument Specialities Co., Inc., Little Falls, New Jersey) at all of the joints. The two systems are grounded separately and are interfaced via opto-isolators whose circuit diagrams are shown in Figure 11. These measured have eli- minated the electrical transient. The dye used is a solution of cresyl-violate perchlorate and rhodamine 6G in methanol which emits at 660 nm, a wavelength close to the absorption maximum of the PS II reaction center (680 nm). The concentration and cresyl-violet to rhodamine ratio of the lasing solution have been determined experimentally to give optimum light intensity at moderate (16 KV) voltages. c. Light Monitor and Filters The light intensity may be monitored during the course of the experiment. The monitor is a glass plate between white surfaces with openings to allow most of the light to pass through. Part of the light is reflected by the plate glass surface and is then detected and integrated by four photodiodes (Hewlett-Packard) connected in “3 .Loumaomwtouao ovoflv Lon amnwmflc uflsonwo .FF aeamfia All ozaomo mug}... 4. 0230mm .2335 1 02.2.... a cam t . c v: e. 85m 350. J Eu 7 FDOJHoIIIJ C. W n 35. - u o .melu. coo. ozaomo mmmfi 08+ 8.... axon» .2 w. oznomo Enema - - t - l ozfcmmam >v~L 4k 1 .1." some .3 0.x— amm N... OF2_.C. 95:49 >m+ odnn Enema oziE >m+ «axow mOquomTOPQO onO “5 parallel with a 820 pf capacitor (Van Best, 1977). The voltage across the capacitor can be read from an oscilloscope (B&K Precision model 1A71B). This circuit is mounted on a filter holder also accomodating neutral density filters. A block diagram showing the main components of the kinetic EPR instrumentation is shown in Figure 12. Because of limited time resolu- tion in the present system, only the pH dependence of signal IIf decay kinetics will be studied. Modification of the lock-in amplifier (Princeton Applied Research model 126) shown in Figure 12 is now in progress which will decrease the time constant from about 800 us to 50 us. This should allow us to perform analogous experiments to study the pH dependence of signal IIv decay kinetics. f 2. Spinach and Chloroplast Suspensions Market spinach is used in this series of experiments. Chloro- plasts are prepared as described in Section I with the following modifications. After centrifugation, the chloroplasts are resuspended in 0.8 M (pH = 8.0) TRIS buffer and left exposed to light for twenty minutes. This procedure is referred to as TRIS washing and results in the release of manganese bound to the thylakoid membrane with the concomitant loss of oxygen evolution. The chloroplasts are centrifuged, washed in the reaction mixture which is 0.” M in sucrose, 10 mM in NaCl, and 50 mM in buffer (MES for pH = 5.5, 6.0 and 6.5, HEPES for pH = 7.0 and 7.5 and TRICINE for pH = 8.0 and 8.5) and finally resuspended in a minimal volume of this mixture. The chloro- plasts are maintained at 0°C during this procedure. 116 .aossasaaaaahmafl mam osptasx Lo aaewase soon .NF acumen 117 ”230.50 I 02.2; d. i cum «modems . macaw motzozFJ >.x .2263 o EoQU c9538 19.—ho \ 10.3.8 I Z. Wutxug 0230mm «undu— lvl $53 In... maoum J U1 .3458 a; ozaomo Em 57mm: nomeouam be mam SQZ<_ O. I F———- 500 ms ——t f C. With 2 x 10"3 M ascorbate. D. With 10"5 M hydro- pH = 7.5 (time constant quinone and 2 x 10"3 = 3 ms, 120 scans averaged) M ascorbate. pH = 7.5 (time constant = 3 ms, 200 scans averaged) 5A .mcowufincoo when» some: moauocfix zmomc adamantfin on» moumnummosoc mean one .nmocou coauooam msocmwoxo no mocmmnm on» :H m.» n ma um mafia .n> :ofiumcpcmocoo MHH Hmcwam mo moa Hmmsumc on» Low moan moaiwaom < .m_ teamsa 55 mqu 54... is .293 e. O. 2 _ ~mL0 1000 500 750 TIME (ms) 250 _ as 4 o 5. 4" :5 mqua 39m 3— .893 a. 30—— 56 Figure 16. A semi-log plot of the natural log of signal IIf concen- tration vs. time at pH = 6.5 with an added ascorbate concentration of 2 x 10'3 M. The plot demonstrates the first order decay kinetics followed under these conditions. 57 4.5 — I 4.0 — . ’1?- . =35 _ . '6 c O .9 3 O E O 3.0 —- . 2.5 ~— I I I A 0 250 500 750 l000 time (ms) 58 Similar plots at the other experimental pH values also reveal that the decay of signal II in the presence of added ascorbate follows f first order kinetics. These observations suggest that at these ascorbate concentrations, signal IIf decay follows pseudo-first order kinetics and that the observed rate constant, kobs’ can be separated into the product of a second order rate constant and an essentially invariant concentration of ascorbate. Sig-5:]- = k [2?] [HAsc] k = k [HAsc] obs To investigate the implication of pseudo-first order kinetics, addi- tional experiments were performed to relate the observed rate of decay of signal IIf to the concentration of added ascorbate. These results, at both pH = 6.5 and 7.5, are shown in Figure 17. The linear dependence at both pH values of the observed rate substantiates the proposed, pseudo-first order kinetic scheme. A third set of experiments was performed using hydroquinone as the exogenous electron donor. Hydroquinone, with a pKa = 10.35, was chosen because it exists almost exclusively in its fully protonated state in the pH range used. This is in contrast to ascorbate (pKa = H.10) which exists largely as an anion above pH 5.5. Figure 14- D is the trace obtained at pH = 7.5 with an added hydroquinone con- centration of 10"5 M. At this low concentration, it was necessary 3 to also add ascorbate (2 x 10- 7M) needed to reduce the hydroquinone after it is oxidized by the signal IIf radical. A further increase 59 Figure 17. A plot of the observed decay rate of signal IIf (k ) obs vs. concentration of ascorbate at two different pH values. 60 obs (5") 7— lmM 5mM lOmM ISmM [ASC] 61 in the rate of signal IIf decay is evident upon addition of hydro- quinone. A semilog plot shows that the decay kinetics are first order. Figure 18 summarizes the data obtained at all pH values in the presence of exogenous electron donors. It is a plot of the decay half-time vs. pH. An overall pattern of pH dependence in the decay kinetics of signal IIf emerges, the reduction of the paramagnetic intermediate being faster at low pH values. D. Discussion 1. Endogenous Electron Donors The results of the analysis of the kinetic traces obtained in the absence of exogenous donors are summarized in Table 1. In each case the decay of signal IIf can be analyzed as the sum of two, distinct decay processes, one fast and another relatively slow. By plotting the natural log of the signal IIf radical concentration vs. time, and by back extrapolating from the end of the time course, ks, the slow rate constant can be determined from the slope and an amplitude, As, that proportion of the total signal following the ks decay rate, can be approximated from the intercept at time zero. After subtracting the contribution made by this process from the overall observed rate, kf, the rate constant for the fast process can be determined. These values are tabulated. Several conclusions can be reached from these data. The ks’ or slow decay rate constants, do not vary as strongly with pH as do the values of kf, which tend to become appreciably faster at higher pH values. A second observation is that, as the pH is increased, a larger fraction of the total signal 62 Figure 18. A plot of signal IIf decay half-times vs. pH for three experimental systems; ( ) with 2 x 10'3 M ascorbate, (o) with 10-5 M hydroquinone and 2 x 10"3 M ascorbate and ( ) with 5 x 10-5 M hydroquinone and 2 x 10'3 M ascorbate. 63 6007— 500— 400 g KAscorbate “£1 300 200ms kmo“5 MHQ lOOms‘ -5 ‘ 5"0 HQ} ' ___._—-——'. M’T’T I L . 5.5 6.0 6.5 7.0 7.5 8.0 8 5 6N Table 1. A breakdown of the bi-phasic decay of signal II in the f absence of exogenous donors; k8 = slow decay rate, kf = fast decay rate, AS a percentage of total signal following slow kinetics, Af : percentage of the total signal following fast kinetics. pH k A k s s f f 8-1 x 8-1 1 5.5 .885 70.A 6.15 29.6 6.0 .9U8 65.2 4.73 3A.6 6.5 .801 7A.5 5.85 25.5 7.0 .814 82.9 5.13 17.1 7.5 .707 80.1 7.96 19.9 8.0 .7AA 86.5 9.U8 13.5 8.5 .727 8A.2 -- 15.8 65 decays with the slower rate constant. Because the nature and concen- tration of the endogenous electron donors giving rise to these kinetics are unknown and experimentally uncontrollable, a more detailed analysis of these data has not been carried out. 2. Exogenous Electron Donors The data presented in Figure 16 demonstrate that the decay of signal IIf, which reflects the reduction of a free radical, ZT, proceeds by pseudo-first order kinetics in the presence of ascorbate. The rate constant calculated for this process is a function of pH (Figure 18). The following kinetic scheme can be used to rationalize these observations: 2? + HAsc 5 z + HAs6' where Z? is the free radical species giving rise to signal IIf and HAsc represents neutral ascorbate. The decay of signal IIf is given by: 'glgfl = k [2?] [HAsc] The concentration of neutral ascorbate is related to the pH by: H” + Asc" .155“ HAsc .__ If this were an accurate description of the kinetics, the observed rates, kobs’ would be proportional to the concentration of neutral ascorbate. Figure 19 is a plot of these two functions. At low pH values the agreement is good but the correlation falls off with increasing pH. This observation suggests a second model to explain the reduction kinetics of Z? as a function of pH: 66 Figure 19. A plot to the concentration of neutral ascorbate (solu- tion 2 x 10"3 M in ascorbic acid) vs. pH (0) and a plot of k0 vs. bs PH (0). 67 -=[HAsc] ° ' kobs 5.5 68 N z. + HAsc .. z + HA36° + -k2 Z. + Asc + Z + Asc' H+ + Asc' -£§e HAsc ,__ In this scheme, both neutral ascorbate and its anion reduce z? and the decay of signal IIf is now given by: + 'QJZ*J : k [HAsc] [2?] + k [Asc-] [ZI] dt 1 2 By introducing the ascorbate equilibrium this becomes: 'th-Z—Ll = k1 [In 1K£A3°-] [21]] + k2 [Asc'] [2?] which reduces to: . k [11*] +1. 31—1 = [2’1 [18°] T At low pH values, the H+ concentration is high and the k1 kinetics will dominate. At high pH values, the 3* concentration is low and the k2 kinetics will dominate. From experiments in which kobs is obtained as a function of ascorbate concentration (Figure 17), the values of k1 and k2 can be calculated. The slopes of these plots can be used to calculate d(kobs)/d[Asc'] which is equal to k1 [H+] /Ka + k . Solving this set of equations leads to k = 1.N x 10” 2 1 ‘MI1 3'1 and k2 = 6.6 x 102M-1 3-1. 69 These values of k1 and k2 have been used to calculate the overall decay rate of signal II as a function of pH and these calculated f rates, kcalc’ subsequently compared to the observed rates, kO bs' Figure 20 is a plot of koala/Robs vs. pH for two sets of experimental data obtained with different preparations of chloroplasts. The relationship is linear in both cases but demonstrates a variable pH dependence. The validity of this kinetic scheme is supported by these results; the non-zero slope, however, is an indication that some subtle pH dependence has not been taken into account. Two such dependencies emerge as possibilities. The first involves the concept of the transmembrane pH gradient or ApH as developed in Section I-D. In the calculation of the kcalc values, the H+ concentration used in the rate expression is taken from the pH of the buffering medium. Based on the model developed in Section I-D, however, the pH inside of the thylakoid membrane will not be the same as that outside unless there is complete un- coupling of phosphorylation. Yamashita and Butler (1969) have shown that upon addition of electron donors, TRIS washed chloroplasts con- tinue to undergo phosphorylation, an indication that a pH gradient develops during exogenous donor oxidation even after the inhibition of oxygen evolution. Using the rate expression, it is possible to calculate the internal H+ concentration necessary to give the observed decay rates and from these concentrations and the pH values of the buffering medium, to further calculate a series of ApH values. These are plotted as a function of external pH in Figure 21. The ApH values obtained by this method are entirely reasonable and their increase 70 Figure 20. A plot of kcalc/kobs for two different chloroplast suspensions. 0.5 — 71 6.5 7.0 7.5 8.0 72 .cocon copuooao msocomoxo am no owom cannoomm m.mlop x m :0“: 9HH Hmcmfim mo mofluocfix swoon oo>nomno on» Bonn nonmasoamo.ma Hmcoouxo .m> ma< no poaq < .PN mgsmHm 73 8.0 7.5 7.0 6.5 6.0 5.5 7n with increasing pH can be qualitatively explained by the model developed in Section I. As Figure 9 illustrates, an uncoupling agent with a pKa that falls in or below the experimental pH range gives rise to a variation in ApH with pH. At least two species with pKa values in this range, TRIS with a pKa = 8.08 and the buffer (MES with a pKa = 6.1, HEPES with a pKa = 7.5 or TRICINE with a pKa = 8.1) are present in the chloroplast suspension and could be acting as weak, pH dependent uncoupling agents. This could also explain the difference observed between the two different chloroplast preparations. The difference in pH dependence may reflect the more efficient washing of one sample to remove TRIS buffer from the suspension. This experi- mental difference could lead to different levels of uncoupling effi- ciency and consequently to different pH dependencies. A second model to explain the pH dependence of the kinetics involves the postulation of a net negative charge on the inside of the thylakoid membrane, an idea which could also help to explain the large difference in the magnitudes of k and k2. The magnitude 1 of k1, which is associated with the reduction of 2? by neutral ascorbate, if twenty times greater than that of k , associated with the reduction 2 of Z? by the ascorbate anion. This suggests that ZI may be more accessible to the neutral form of the reducing agent than to its anion. This could be explained by a net negative charge on the inter- nal membrane surface. Support for-the presence of this surface charge comes from the electrophoretic experiments conducted by Nakatani SE El- (1978) which demonstrate the presence of a net negative charge on the exterior of the thylakoid membrane. They studied the effect of pH on the mobility of the thylakoid membrane when placed in a 75 conducting solution in the presence of an electrical field. When the suspension pH is greater than “.3, the thylakoids move toward the anode, demonstrating that they carry a net negative charge. Below pH values of n.3, the direction of electrophoretic migration is reversed. These results were interpreted to indicate that there exist, on the exterior surface of the thylakoid, groups with a pKa of about “.3. If the internal surface of the thylakoid carries a similar charge at neutral pH, the following scheme might explain the pH dependence of signal IIf decay. As the pH of the suspension medium, and therefore of the thylakoid interior, decreases, an in- creasing fraction of the surface negative charges, possibly arising from carboxyl groups or pigment-protein complexes of the antenna chlorophyll (Nakatani gt al., 1978), would protonate, leading to a decrease in the surface charge. This would result in an effective increase in the magnitude of k the rate constant assocated with 2. the anionic reducing agent, since the ascorbate anion would be less strongly repelled by the membrane as the net negative charge on it surface was decreased. Good fits to the experimental data are ob- tained for values of k2 which range between about 2200M-1 s-1 at pH = 5.5 to 800M'1 3'1 at pH = 8.5. Further experiments by Nakatani 733,31. (1978), however, suggest that this explanation of the pH dependence of signal IIf decay may be unlikely. Between pH values of 6.0 and 10.0, the rate of electrophoretic migration of the thyla- koid is found to be independent of pH, indicating no change in the magnitude of the exterior surface negative charge in this pH range. However, the experiments of Itoh (1978) on ferricyanide reduction demonstrate that the negative charge on the exterior of the thylakoids 76 can at least be masked, if not neutralized, by variation of the suspen- sion pH, as discussed in Section I-D. All of these experiments reflect the nature of the exterior surface of the thylakoid membrane and whether the interior exhibits a similar net charge or will behave analogously to variations in pH remains to be tested. Experiments are planned to test both of these models. A study will be made of signal IIf decay kinetics in the presence and absence of ammonium ion. If the addition of this known uncoupling agent is found to depress the pH dependence, presumably through the dissi- pation of the ApH across the membrane, support would be gained for the first model. The second model will be tested by studying the pH dependence of signal IIf decay upon addition of nigericin and variation of salt concentration. Nigericin selectively permits the incorporation of potassium ions into the thylakoid compartment (Shavit et al., 1968). A variation in the pH dependence with potassium ion concentration could demonstrate the shielding of negative surface charges by the cation, with a concomitant increase in the magnitude of k2. An additional set of experiments, those involving hydroquinone as the exogenous electron donor, substantiates the placement of Z? in an environment more easily accessible to neutral than to charged species. Hydroquinone, with a pKa of 10.3A, was selected as an electron donor because over the experimental pH range, it exists almost exclusively in its neutral form. The used of relatively low concentrations of hydroquinone, 10-5 M, necessitated the addition of ascorbate to reduce the hydroquinone oxidized by ZT. The use 77 of hydroquinone substantially decreases the half-time of signal IIf decay as illustrated in Figure 18. The decay of Signal IIf in the presence of hydroquinone follows pseudo-first order kinetics, which allows the computation of a hydroquinone rate constant, kHQ’ from the relationship k0 kHQ [HQ]. At pH = 5.5, the calculated kHQ bs = S M-1 -1 = 9.59 x 10 s . As a comparison of the relative efficiency of neutral ascorbate and hydroquinone as reductants of ZI, this kHQ can be compared to k1 from the ascorbate case at pH = 5.5, when the k1 term is dominant and the kinetics reduce to k0 k1 [HAsc]. u 1 8-1. bs The value of k1 under these conditions is about 7.75 x 10 35 A comparison of these rate constants shows that hydroquinone is about ten times more efficient than ascorbate in reducing 2?. This may indicate the situation of Z? in a highly non-polar environment but additional work is required before such an assignment may be made. The studies described have provided information about the role of the thylakoid membrane in the chemical reactions of plant photo- synthesis. The results and the models derived from them contribute to the understanding of the function of the membrane in photosynthetic oxygen evolution and emphasize the importance of this role. BIBLIOGRAPHY BIBLIOGRAPHY Babcock, G. and Sauer, K. (1973) Biochim. Biophys. 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