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' ‘.- 51> I 'I ‘ -l n ' *“ink . J ” I" ‘,-'I ' l " " 4‘.“ | A: _ "‘L ‘. n J ’4'7 -‘ f.” III“. "I' ' I \.'.‘»“‘I.."‘ ."(N-Iu'a' Lu‘hu" u- ..'. . -‘ Hm” ‘ ILI IL" 3' .' - .I L I.” .f"';:1""'I;I“n :‘: 23hr." "; '1" H J -' II»! \I I I:.I"..;:‘I‘ 2 v“ {‘(J‘r'f'alr . .. L UN)»- ‘La ‘L .L.' ' --L’ ‘ L‘s .. 1N1".",'.VJL.3;'_uJIj 3);? . " ”-‘b‘t‘. 3% -- J“ "n 13 PIX“ I .L L‘"L L <105; b) pokeweed TSF-IIa partigles (0.69 mg Chl/ml), instrument gain==0.8><10 ; c) Chlamydomonas PSII particles é0.53 mg Chl/ml), instrument gain = 1.25><10 ; d) Chlamydomonas PSII DEAE particles (0.44 mg Chl/ml), instrument gain = 1)<106. A modulation amplitude of 4 G and a time constant of 0.2 s were used in recording all spectra. (From ref. 92). . . . . .31 Signal II kinetic transients as a function of magnetic field in Chlamydomonas DEAE PSII particles. The spectrum of Signal II recorded in the dark is shown in (a); field ‘Vi" Figure Page positions at which the kinetic traces shown in (b) were recorded are indicated as points A, B and C on the spectrum. Field point D is the zero crossing of Signal II. For the spectrum, the following instrument parameters were used: 0.2 s time constant, 200 s sweep, 4 G modulation amplitude, 20 mW microwave power. For the kinetic traces, 200 flashes, given at a rate of 0.25 Hz, were averaged, the instrument time constant was 500 us. DEAE particles containing 0.44 mg Chl/ml were used. . . . . . . 38 Effect of donor addition on Signal II decay in Chlamydomonas DEAE PSII particles. The decay of Signal II in DEAE particles (0.44 mg Chl/ml) without (a) and with (b) the addition of 40 uM BZ is shown. Each trace is the average of 200 flashes, given at a frequency of 0.25 Hz. Instrument conditions: 0.5 ms time constant, 4 G modulation amplitude, 20 mW microwave power, field set at field point A (see Fig. 6). In (c), the BZ concentration dependence of the observed decay rate constants is summarized. . . . . . . . . . . . . 41 Signal II kinetic transients in different PSII reaction center preparations. In (a) and (b), Chlamydomonas particles (0.39 mg Chl/ml) were used. No further additions were made in (a), overall decay halftime = 36 ms (see text); in (b) 40 uM B2 was added as an electron donor, decay halftime = 5 ms. Each trace is the average of 200 flashes, the instrument time constant was 200 us. In (c) spinach TSF-IIa particles (0.78 mg Chl/ml) were used and 40 uM B2 was added as an electron donor. The decay halftime was 30 ms, the instrument time constant was 500 us and 150 flashes were averaged. In (d), (e) and (f), pokeweed TSF-IIa particles (0.39 mg Chl/ml) were used. In (d) no further additions were made, decay halftime==170 ms; in (e) 20 uM BZ was added, decay halftime==45 ms; in (f) 40 uM BZ was added, decay halftime = 22 ms. For these traces, an instrument time constant of 200 us was used and 255 flashes were averaged. In traces (a)-(f), -vii- Figure 10. 11. Page a modulation amplitude of 4 G and micro- wave power of 20 mW were used. All kinetic traces were recorded at field setting A in Fig. 6. . . . . . . . . . . . . . . . . . . . . 43 The effect of lipophilic anions on the decay of Signal II in Chlamydomonas particles PSII and DEAE PSII particles. In (a) and (b) Chlamydomonas particles (0.44 mg Chl/ml) were used. In (a) 20 uM TPB was added (decay halftime = 37 ms) and in (b) 5 uM ANT 2p was added (decay halftime = 23 ms). Instrument conditions: 200 us time constant, 200 scans averaged. In (c) DEAE particles (0.40 mg Chl/ml) to which 5 uM ANT 2p was added were used. The decay halftime is 95 ms. Instrument conditions: 500 us time constant, 200 scans averaged. Other settings as in Fig. 8. . . . . . . . . . . . . .48 The effect of cations on the decay kinetics of Signal II in tris-washed chloroplasts. Ascorbate (2 mM) was used as an exogenous donor. a) No further addition; b) 200 mM KCl; C) 50 mM MgClz; d) 50 mM CaClz; and e) 50 mM SrCl . A time constant of 1 ms was used in all tfie experimental traces which are each the average of 120 flashes given at a repetition rate of 0.25 Hz. The decay halftimes (tg) measured from the experimental traces are: a) 350 ms; b) 290 ms; c) 285 ms; d) 94 ms; and e) 85 ms. . . .50 The effect of cations on the decay kinetics of Signal IIf in tris-washed chloroplasts in the presence of neutral donors. a) 0.4 mM DPC; b) 0.4 mM DPC and 50 mM CaClz; c) 0.4 mM DPC and 50 mM SrClz; d) 0.4 mM DPC and 50 mM MgClz; e) 30 uM benzidine and 2 mM ascorbate; f) 30 uM benzidine, 2 mM ascorbate and 50 mM CaClz; and g) 30 uM benzidine, 2 mM ascorbate and 50 mM MgC12. A time constant of 1 ms was used in a-d and 0.5 ms in e—g. The experimental traces in a-d are the average of 120 flashes given at a repetition rate of 0.25 Hz, whereas traces in e-g are the average of 150 flashes given at the same rate (0.25 Hz). The decay halftimes (tk) measured from the kinetic traces are: -viii- Figure 12A. 128. 13. 14. 15. Pages a) 64 ms; b) 60 ms; c) 60 ms; d) 104 ms; e) 16 ms; f) 16 ms; and g) 29 ms. . . . . . . . 53 Effect of the dark time (td) between flashes on the magnitude of Signal IIf [A==(AIIf) /(A¥§¥), where AIIf is the measured max amplitude and AIIf is the amplitude i3 the absence of DCMU]. 3 mM Fe(CN)6‘, 20 mM Mg++ and 100 uM DCMU wefe added to a suspension of tris-washed chloroplasts at pH 7.6. The instrument time constant was 1 ms; 150 flashes were averaged. The dashed lines show the theoretical fit of these data according to Eq. (3.8). . . . . . . . . . . . . . . . . . 62 Graphical determination of the apparent rate constant of QA reoxidation by ferricyanide. . . . . . . . . . . . . . . . . . 64 Effect of Mg++ concentration (MgClz) on the magnitude of Signal IIf. 3 mM Fe(CN)%' and 100 uM DCMU were added to a suspension of tris-washed chloroplasts at pH 7.6. The instrument time constant was 1 ms; 150 flashes were averaged at a frequency of 0.33 Hz (td==3 sec). . . . . . . . . . . . . . .67 Effect of ferricyanide concentration on the magnitude of Signal IIf. The amounts of ferri- and ferrocyanide shown above were added to a suspension of tris-washed chloroplasts at pH 7.6, which contained 40 mM Mg++ and 100 uM DCMU. The redox potential in all samples wasl~460 mV. 150 flashes were averaged at a frequency of 0.5 Hz and with an instrument time constant of 1 ms. . . . . . . .'. . . . . . . . 69 Effect of negatively charged species on 'Signal IIf in tris-treated DCMU—inhibited chloroplasts, pH 7.6, in the resence of 40 mM Mg+ and 6 mi_4 Fe(CN) ‘. a) No furthe addition, td==3.5 sec; b) 60 mM Fe(CN) ', t =3.5 sec; 9) 60 mMFe(CN)é", td==8 sec; 3) 200 mg 80; (Na2804), td==3.5 sec and e) 200 mM 803, td==8 sec. The redox potential in (a) was 582 mV, in (b) and (c) 405 mV and in (d) and (e) 566 mV. The instrument time constant was 1 ms and each trace is the average of 150 flashes. . . . . . . . . . . . . . . . . . .72 -ix- Figure 16. 17. 18. 19. Page Phenazinemethosulfate (PMS) as an artificial acceptor in tris-treated DCMU-inhibited chloroplasts at pH 7. 6. a) 1 mM PMS; redox otential 260 mV; b) 1 mM PMS and 1 mM Fe(CN) ; redox potential 520 mV. 150 flashes were averaged at a frequency of 0.25 Hz. The instrument time constant was 200 us in (a) and 1 ms in (b). . . . . . . .78 The effect of donor addition on the decay of Signal IIf in tris-washed chloroplasts. In (a) 2 mM sodium ascorbate was added as a donor: the observed rate constant for Signal IIf decay is 4. 2 3’1; in (b) 100 uM hydroquinone and 2 mM sodiumk ascorbate -1 comprised the donor system, -—16. 4 s in (c) 200 uM hydroquinone and b3 mM sodium ascorbate were used, kobs -=34 s 1. The instrument time constant in (a) was 1 ms which was decreased to 500 us in (b) and (c). Bach trace is the average of 180 flashes given at a frequency of 0.25 Hz. . . . .92 o I The effect of ANT 2p on the decay kinetics of Signal IIf in tris-washed chloroplasts. ANT 2p at the indicated concentrations was used in (a), (b) and (c). A time constant of 500 us was used in all three experimental traces which are each the average of 180 flashes given at a repetition rate of 0.25 Hz. In (d), a semilog plot of the decay of Signal IIf in (b) is presented. A hand—drawn curve was fitted to the data and the amplitude of Signal IIf at various times was extracted from this curve. . . . . . . . . . . .95 The effect of picrate addition on the decay kinetics of Signal IIf in tris-washed chloroplasts. In (a)-(e) 9 mM K3Fe(CN)6 and 40 mM CaClz were added to a suspens1on of tris-washed chloroplasts at pH 7. 6 and picrate at the indicated concentrations was added. Note that the time axis is compressed by a factor of 2 in (a) compared to the other traces in the figure. In trace (f), the K3Fe(CN)6 and CaClZ were omitted from the reaction mixture which was 180 uM in picrate; in (g) 9 mM K3Fe(CN)6 and 180 uM picrate were present but the CaCl was omitted. The instrument time consgant was 500 us; 150 flashes were averaged at a frequency of 0.25 Hz. . . . . . . 98 -X— Figure 20. 21. 22. Page Graphical determination of the second-order rate constants for the decay of Signal IIf in the presence of various ADRY reagents. The observed rate constant for the decay of Signal IIf in the presence of the indicated ADRY reagent is plotted against the corresponding ADRY concentration. The slope of the straight lines which results is the second-order rate constant. These are tabulated in Table IV. . . . . . . . 100 The effect of an ADRY reagent in conjunction with an electron donor system on the decay kinetics of Signal IIf. In (a)-(c) the ADRY, CCCP, and the donor system, hydroquinone/ascorbate, were used. The CCCP concentration in (a) and in (c) was 50 uM; no CCCP was present in (b). Hydroquinone (150 uM) and sodium ascorbate (2 mM) were added in (b) and (c), but were absent in (a). A time constant of 1 ms was used for traces (a)-(c), each of which is the average of 150 flashes given at a repetition rate of 0.25 Hz. In traces (d)-(h) the ADRY, ANT 2p, and the hydroqui- none/ascorbate donor system were used. In (d) and in (f), the hydroquinone concentra- tion was 20 uM and ascorbate was present at 2 mM; the donor system was omitted in the other traces. The ANT 2p concentration in traces (e)-(h) was 10 uM; it was omitted from the chloroplast suspension used in obtaining trace (d). In (9), 12 mM K3Fe(CN)6 and 40 mM CaClz were present and in (h) 40 mM CaCl was added. An instrument time constant of 500 us was used in obtaining the experimental data in (d)-(h). 180 flashes, given at a rate of 0.25 Hz, were averaged to obtain the final decay curves. . . . . . . . . . . . . . . . . . . . .107 The effect which conditions on the acceptor side of Photosystem II exert on the decay kinetics of Signal IIf in the presence of an ADRY reagent. The decay of Signal IIf was recorded in tris-washed chloroplasts in which the final concentration of added reactants was as follows: (a) 100 uM DCMU, 12 mM K3Fe(CN) , 40 mM CaClz; (b) 100 uM DCMU, 12 mM K Fe(CN) 40MMCaC12, 10 UM ANT 2p: (c) 1 K§e(CN)5 40 mM CaClz, 10 uM ANT 2p; (d) 180 uM DCMU, 10 —uM ANT 2p. -xi... Figure 23. 24. 25. 26. 27. Page For each trace, 180 flashes were averaged at a flash repetition rate of 0.25 Hz. The instrument time constant was 1 ms. . . . . . . 111 The effect of pH on the decay of Signal IIf in tris-washed chloroplasts in the presence of ANT 2a. In (a) tris-washed chloroplasts were resuspended in a buffer which contained 0.4 M sucrose, 0.01 M NaCl and 0.05 M MES. The pH was 5.8 and 15 uM ANT 2a was present. In (b) a buffer consisting of 0.4 M sucrose, 0.01 M NaCl and 0.05 M HEPES at pH 8.2 was used. The ANT 2a concentration was 15 uM. Each trace is the average of 180 flashes, 0.25 Hz repetition rate and 1 ms time constant. . . . .114 Graphical determination of the second- order rate constant for the decay of Signal IIS in oxygen-evolving chloroplasts in the presence of CCCP. The data from Fig. 3 in ref. 146 were used to obtain the pseudo-first order rate constant, kobsr for the decay of Signal IIS at various CCCP concentrations. This rate constant is plotted against the correspond- ing CCCP concentration in order to determine the second-order rate constant as the slope of the straight line which results. . . . . . .122 A model for the role of ADRY reagents in destabilizing oxidizing equivalents generated in PSII. The solid arrows denote proposed reactions in oxygen- evolving chloroplasts, dashed arrows indicate proposed reactions in tris- washed chloroplasts and the dotted lines indicate inhibitor action. . . . . . . . . . . 131 Effect of NHZOH concentration on the 02 rate of PSII preparations at pH 6.0. PSII preparations (10 ug Chl/ml) were suspended in the polarograph ve sel with the exogenous acceptors, Fe(CN) - (3.5 mM) and 2,5-dichloro-p-benzoquinone (250 uM). Addition of NHZOH was followed by immediate illumination to determine the rate of 02 evolution. . . . . . . . . . . . . . . . . . . 137 Kinetic tra sient of the P280 EPR signal. 1 mM Fe(CN) , 1 mM Fe(CN)" and 2 mM NHZOH were added to a suspension of PSII —xii- Figure 28. 29. 30. 31. 32. 33. 34. Page particles at pH 6.0. The instrument time constant was 50 us; 150 flashes were averaged at a frequency of 0.1 Hz. . . . . . . 139 Shape of Signal PEBO (solid line) in PSII preparations obta1ned by kinetic experiments at the indicated field values. The shape of Signal IIs is also shown (dotted line). Insert: Saturation properties of Signal P380 in PSII preparations at pH 6.0. Each p01nt represents the amplitude of the P280 EPR signal at the indicated microwave power. The conditions were the same as those in Fig. 27. . . . . . . . . . . . . . . .141 A model for the inhibitory role of NHZOH in Photosystem II. . . . . . . . . . . . . . . 145 Kinetic transients of Z? at room temperature in NHZOH extracted chloroplasts at pH 7.5; a) no further addition, b) 4 mM ascorbate and c) 50 uM benzidine, 4 mM ascorbate. . . . .153 Graphical determination of the second order rate constants for benzidine (---) and hydroquinone (-—-) donation to Z- in NHZOH extracted chloroplasts. . . . . . . . . . . . .155 Kinetic transients for 2+ at room tempera- ture in NH3 (200 mM) treated chloroplasts at pH 7.5; a) no further addition, b) 5 mM CaClz and c) 200 uM benzidine, 4 mM ascorbate. Time constant==l ms, 250 scans averaged at a rate of 0.25 Hz. . . . . . . . . 158 Kinetic transients for 21 at room tempera- ture in high salt (2 M NaCl, pH:6.0) treated PSII particles at pH 7.5. An equimolar mixture of ferricyanide and ferrocyanide (3 mM each) was used as an acceptor system. a) No further addition, b) 50 uM benzidine, c) 200 mM NH3 and d) 200 mM NH3 and 100 uM benzidine. Each kinetic trace is the average of 200 flashes. Time constant==0.5 ms and the dark time between flashes td==5 sec. . . . . . . . . . . . . . . 161 Kinetic transients for 21 at room tempera- ture in high salt (2 M NaCl, pH:6.0) treated PSII particles at pH 7.5. An equimolar mixture of ferricyanide and ferrocyanide (3 mM each) was used as an acceptor system. a) No further addition, t ==6 sec, b) 15 uM benzidine, td==6 sec, c) 30 uM benzidine, td==6 sec, d) no addition, -xiii- Figure 35. Page t ==2 sec, e) 15 uM benzidine, td==2 sec, and f) 30 pH benzidine, t ==2 sec. Each kinetic trace is the average of 250 flashes. Instrument time constant==0.5 ms. . . . . . . .163 Model for polypeptide and manganese location in the oxidizing side of Photosystem II. . . . 172 -xiv- ASC ADRY ANT 2a ANT 2p ANT 25 B2 CAPS CCCP DCMU DPC EPR FeIIICN (or FeCN) FeIICN FCCP HEPES HZQ (or HQ) MES OEC PMS PS I PS II TPB Tris ABBREVIATIONS sodium ascorbate Accelerator of the Deactivation Reactions of the water-splitting system, Y. 2-(4-chloro)anilino-3,5-dinitrothiophene 2-(3-chloro-4-trif1uoromethy1)anilino- 3-5,dinotrothiophene 2-(3,4,5-trichloro)anilino-3,5— dinitrothiophene benzidine 3-cyclohexy1amino-1-propanesu1fonic acid carbonylcyanide-m-chlorophenylhydrazone 3-(3,4-dichlorophenyl)-1,1-dimethylurea diphenylcarbazide electron paramagnetic resonance potassium ferricyanide potassium ferrocyanide carbonylcyanide-p-trifluoromethoxyphenyl— hydrazone N-2-hydroxyethy1piperazine-N'-2-ethane- sulfonic acid hydroquinone 4-morpholinoethane sulfonic acid oxygen evolving complex phenazine methosulfate Photosystem I Photosystem II tetraphenylboron Tris(hydroxymethyl)aminomethane CHAPTER I . INTRODUCTION In 1771 Joseph Priestley showed that a mouse could not live in a container in which a candle flame had been allowed to burn out, but that a spray of mint restored the air so that after a few days a candle could burn again, or a mouse could live for a time. Thus Priestley showed that a plant could produce oxygen, or, in the language of his time, could dephlogisticate the air. In 1782 Senebier pointed out that plants need carbon dioxide to "oxygenate" the air and fourteen years later Ingenhousz suggested that this carbon dioxide was the source of all the organic matter in plant. In the middle of the nineteenth century Mayer suggested that the light energy was stored, in part, as chemical energy in the organic matter and in the early twentieth century the role of chlorophyll in photosynthesis was known. Today, two centuries after Priestley's experiment, it is well known that of all the ways in which life interacts with light the most fundamental is photosynthesis, the biological conversion of light energy into chemical energy. The overall chemistry of photosynthesis can be expressed in a simple equation. Six molecules of carbon dioxide are -2- taken up from the environment together with six molecules of water. In the presence of light these molecules are converted into a single molecule of glucose and six molecules of 02 are released: 6C0 +6Ho—l‘l-+CH 0 +60 2 2 6 12 6 2 (1'1) For purposes of analysis, photosynthesis is most conveniently divided into two steps: a light reaction and a dark reaction. The light reaction captures energy from the sun in two comparatively unstable molecules: ATP and NADPH. In the dark reaction ATP and NADPH supply the energy needed to form glucose from C02. Both of these reactions, the light and the dark, take place in regions of the plant cell known as chloroplasts. In chloroplasts the photosynthetically efficient photochemistry occurs at a limited number of sites in the pigment-containing membranes. These sites, so-called reaction centers, have a well-defined organization and positioning in the membranes. A reaction center contains a specialized chlorophyll a molecule, which, following electronic excitation, acts as the primary electron donor. At this point it is well known that there are two types of reaction centers in Oz-evolving photosynthetic organisms: the Photosystem I (PS-I)reaction center and the Photosystem II (PS-II) reaction center. These are present in a nearly 1:1 ratio and function in series to drive electrons from the Oz-evolving complex to NADP+ [1]. -3- A schematic representation of the light driven reactions is presented in Figure 1. As shown in this scheme, the reaction center of PS-II extracts electrons from a very oxidizing species and operates at high potential. By contrast, the reaction center of PS-I produces strongly reducing products and thus operates at a low potential. The reaction centers represent only a small fraction of the total pigments and the function of light absorption is fulfilled by other pigments which constitute the antenna system. Antenna pigments transmit their electronic excitation energy, resulting from light absorption, to the reaction centers, via both exciton and Forster transfer [2]. In general there are about 400 chlorophyll molecules per PS-I or PS-II reaction center [3L A study of the flash induced difference spectrum indicates that photoexcitation of Photosystem II involves the bleaching of a special chlorophyll-a, P680' with characteristic negative peaks around 430 and 690 nm [4]. and the formation of a plastosemiquinone anion radical with a positive peak in the absorption difference spectrum around 320 nm, Q- [5]. When the time resolution for A measuring changes of Optical absorbances was improved, the role of a pheophytin molecule as an intermediate acceptor between P680 and QA’ emerged [6]. A second quinone, QE [7], a two electron acceptor, connects QA with the plastoquinone pool which in turn funnels electrons towards Photosystem I. The reduction of P280 leads to the FIGURE 1. Flow of electrons from water to NADP (Z scheme). Abbreviations: {Sln,the oxygen evolving complex; Z, the primary donor to P680; P680' the PS-II reaction center; I, pheophytin, the primary acceptor of PS-II; Q the A! primary stable acceptor of PS-II; Q the secondary acceptor BI of PS-II; PQ plastoquinone pool; cyt f, cytochrome f; pool’ Pcy, plastocyanin; P700, the PS-I reaction center; X, the primary acceptor of PS-I; Fd, ferredoxin, Fd-NADP, ferredoxin NADP reductase. ONIN +IV +NOll\ Nlefimw . oew\\ . m 0+ ooknr 1 V.O+ >UQ/ Ema; On. In . 6,46 x! - 00 Slow H {/1774 H A: 69.2, \E . $042-6“; .. v 0.. a, I x 1 H L QC: -6- formation of a strong oxidant, Z+(E;r~+l.l V, [8]), possibly a plastoquinone cation radical [9], which through the oxygen evolving complex (S-states), oxidizes water to molecular oxygen. In an analogous manner, when light is absorbed by Photosystem I charge separation between the primary donor P700 and the primary acceptor X occurs. In the dark, following the photoexcitation of the reaction center, P;00 is rereduced by electrons transferred from Photosystem II whereas the reduced form of the primary acceptor is reoxidized by an iron sulfur center which in turn reduces NADP. As the result of flash spectroscopic and polarographic studies of the microscopic phenomena, the photosynthetic electron transport from water to NADP has been inferred to be arranged in a zig-zag scheme [10], as shown in Fig. 2. Measurement, via the electrochromic effect, of the rise kinetics of the laser-flash-induced formation of an electric potential gradient across the thylakoid membrane [11] has led to the conclusion that the reaction center complexes span the membrane anisotropically with both primary donors, P680 and P700, located near the inner side and the corresponding acceptors towards the outer side. From Fig. 2 it is apparent that electron flow from H20 to NADP results in a development of both charge (Aw) and proton (ApH) gradients across the membrane. These two components make up what Mitchell FIGURE 2 . Patterns of electron and proton transport in relation to the thylakoid membrane. \Ev Iaoqzm + -9- called the protonmotive force, pmf: pmf(volts) = 0.06(ApH)-+Aw which provides the energy necessary for ATP synthesis [12]. The formation of a molecule of oxygen from water requires the removal of four electrons; at pH 7.0 the average oxidation-reduction potential of these four equivalents is +0.81 V 2H20——+02-+4H+-+4e (1.2) Even though a considerable amount of knowledge has accumulated concerning the process of 02 evolution, the photosynthetic photooxidation of water remains to be elucidated. A good deal of insight has been obtained by considering possible mechanisms for cleavage of water at the oxidizing side of Photosystem II. Upon excitation of PS-II reaction center, the reaction center chlorophyll molecule P680 is oxidized and simultane— ously. Q the primary electron acceptor of PS-II, is A’ reduced. In the dark following the excitation, P680 is reduced by the secondary donor, Z. The subsequent steps in the scheme shown below involve reduction of Z+ by Sn’ the charge-accumulator which carries out the actual oxidation of water, and reoxidation of the primary acceptor QA by the plastoquinone pool. _1 0- ZPQ (Ar—h—V-—>ZP+Q’ (A)—~+Z+PQ-(A/)——\ZPQ- (A>——~>ZPQ (A') Sn Sn+1 (1.3) In 1967 Joliot and co-workers [13] observed that the O2 flash yield in a series of successive flashes varied with a period of four. In other words, the photosynthetic apparatus stores the oxidizing equivalents produced by four flashes and then breaks down two molecules of H20 and produces one molecule of 02. The results of such an experiment are shown in Fig. 3. The model proposed by Kok et al. [14], in order to explain the above periodicity is shown in Fig. 4. According to this model each 02 center undergoes the cyclic series of reactions shown in the scheme. The states of the centers, Sn' differ by the number of oxidizing equivalents, indicated by n. To fit this model to the experimental data, two assumptions were made: (1) During each flash, a fraction (a) of the 02 centers does not undergo a phototransition ('misses') and another fraction (8) undergoes a double transition (S-———+Sn+2 'double hits'). n (ii) The S1 state and SO state are both stable in the dark. Since the S and 83 states have rather short 2 lifetimes, the distribution of S states after about 15 m1nutes dark 13 So:Sl:Sz:S3 = 0.25:0.75:0:0. -11- FIGURE 3. O2 flash yield sequence observed with isolated chloro- plasts after a long dark period. (Ghanotakis, D.F. and Buttner, W.J., unpublished data). -12.. ”$0232 13... ON or or n o mmmsaz :mfiu .m> ed: No wit/Be T l I l l [ T l l T— I 0.0 0:0. 04. SSA/“A FIGURE 4. Schematic by Kok et oxidation represent represent -13.. diagram of the model of 02 evolution proposed a1. [14]. The subscripts (n==0-4) denote the state of the 02 system. The steps Sfl_—+S: phototransitions and the steps s;——+Sn+1 the subsequent dark relaxations. -l4- 2:420 s by S4 o\,\/ a: ,/ s. 33 \ /“ by S3\ 5T * Sii‘t-‘~_____——’ SE:’//// -15- Although the chemical identity of the charge collector (Oxygen Evolving Complex, OEC) has not been established, indirect evidence suggests that some form of thylakoid- bound manganese is involved. Depletion of manganese in plants or algae by withholding it from the growth medium leads to the loss of 02 evolution capacity [15]. Activity can be restored upon readdition of Mn2+ to the growth medium. Various experiments point to a site on the donor side of Photosystem II as the location for the manganese requirement [16,17]. Manganese occurs in several pools in higher plant or algal cells. A portion of manganese occurs as aqueous Mn2+ and gives rise to an EPR signal characteristic of hexaaquomanganese (+2). It can be removed completely by cell rapture and washing of the pigment membranes with chelating agents such as EDTA [18]. Yocum et al. [19], using EPR spectroscopy, found that chloroplast thylakoid membranes isolated in the presence of EDTA retain high 2+ rates of 02 evolution but contain no Mn that is detectable by EPR at room temperature. The total Mn2+ content of these preparations is 4.6 per 400 chlorophylls; 0.6 Mn2+ can be released by addition of Ca2+, a treatment that does not affect 02 evolution. The remaining Mn2+ appears to be functionally associated with 02 evolution activity. Inhibition by Tris, NHZOH, or heat will release a small fraction of Mn2+ from these membranes (=z25% with -l6- Tris, for example). Addition of Ca2+ further enhances Mn2+ release so that for Tris and for NHZOH, 2 and 3, 2+ per 400 chlorophylls are extracted respectively, Mn from the OZ-evolving complex. Dismukes and Siderer [20] reported the observation of a multiline ESR signal in spinach chloroplasts given a series of laser flashes at room temperature and rapidly cooled to -l40°C. The spectrum was attributed to a pair of antiferromagnetically coupled Mn ions, or possibly a tetramer of Mn ions, in which Mn(III) and Mn(IV) oxidation states are present. This manganese complex was identified with the 82 state of the OEC. In the process of H20 oxidation four protons are released for every molecule of O2 liberated. One might expect that these protons would be released concurrently with the 02, as suggested in the early scheme of Kok et al. [14]. The experimental measurements show that this does not happen. Although the detection of proton release associated with water oxidation is complicated by other proton translocations across the thylakoid membranes that interfere and therefore must be subtracted out [21,22], each of the three groups that has carried out such studies agrees that the concerted process cannot be correct [22-24]. For the present the data do not justify going beyond the simplest whole-number values for the proton release pattern. Even so, there remains a disagreement between Fowler [22] and Saphon and Crofts [23], -17- on the one hand, who believe that the pattern is l,0,l,2 for the number of protons released, respectively in the steps 80+51, 81+82, 82+83 and S3+{S4}+SO, and Junge et al. [24] who believe that the pattern is 0,l,l,2. The experimental results of these groups clearly differ, especially with respect to the release of protons following the first flash and the amplitude of the oscilla— tions of period 4. Although it is generally assumed that protons are released directly from H20, it is possible that protons are released from a protein in the OEC. It has been observed for hemoglobin that there is linkage between the binding of oxygen and the binding of protons by the protein. The oxygen-linked ionizable groups change pK upon addition or removal of oxygen, thereby exchanging protons with the solvent (the so-called Bohr effect) [25,26]. Further investigations of these measurements and the correlation of their kinetics with electron trans- fer kinetics are needed in order to establish the number and the origin of protons released in a series of flashes. The close coupling between the reaction center and the oxygen evolving complex (OEC) can be disrupted by various physical and chemical treatments resulting in rather selective inactivation, or 'decoupling' of the OEC [4,27,28]. Exposure of isolated thylakoid membranes to Tris [29-31] or to NHZOH [32-35] represent mild procedures for inactivation of the oxygen evolving reaction system. After the above treatments the decoupled -13- PS-II reaction center, though incapable of H20 photooxida- tion, will still photooxidize a wide variety of chemically unrelated artificial electron donors, for example, ascorbate [27,36]. Apparently, the system after the treatments remains photochemically active, since the photooxidation of artificial electron donors via PS-II and PS-I proceeds with high quantum efficiency [33,37]. Among the compounds that have been known to inhibit oxygen evolution acting at the oxidizing side of PS-II are various amines, for example ammonia [38,39] and methylamine [39]. The inhibition by ammonia and methylamine appears to be rather direct and does not result in the release of bound Mn that occurs with higher concentrations of NHZOH and high concentrations of Tris. Velthuys [40] and Frasch and Cheniae [31] proposed that water binds to manganese in the OEC by a Lewis acid-Lewis base mechanism and that amines, being better Lewis bases, are able to compete with H20 for binding sites. Other treatments which result in inhibition of 02 evolution at the oxidizing side of PSII are Cl- depletion from chloroplasts [29,41] and extraction of certain polypeptides by using various techniques [41,42]. Izawa et a1. [39] provided firm evidence for a specific function of C1- in reactions closely associated with 02 evolution itself. Using mild techniques to deplete C1- from normal Cl--containing chloroplasts, they obtained preparations which, upon addition of Cl- (~5 mM), showed a 4- to lO-fold -19- increase in rates of 02 evolution. The role of two polypeptides, the 17 and the 23 Rd, has also been studied extensively in a series of extraction-reconstitution experiments [41,42]. A. Experimental Approaches The experimental techniques which have been used to unravel the details of photosynthesis include such spectro- scopic techniques as visible and UV absorption changes upon photoexcitation, prompt and delayed fluorescence (Luminescence), nuclear magnetic resonance (NMR) and elec- tron spin resonance (ESR). Various biochemical approaches, for example, extraction of certain polypeptides followed by selective reconstitution, have also been used. A summary of the various spectroscopic techniques is given below: 1. Optical and UV Changes A primary use of optical spectroscopy is to charac- terize different molecular species or transient states and to follow in viva their modifications during the photosynthetic process. Fast spectroscopic studies have provided a great amount of data on the primary reactions. D6ring et a1. [4] obtained the absorption difference spectrum of P which showed major bleaching at 682 680 and 435 nm. The authors attributed the spectrum to a "sensitizer" which they designated chlorophyll a but II’ Butler [43] proposed, in a model which is now generally -20- accepted, that it is due to the oxidation of P680‘ Absorption transients attributed to P680 include a small band at 820 nm [44], similar to that of a chlorophyll a cation-radical. This absorption band is very useful for kinetic studies, permitting relatively straightforward observation of P280 with excellent time resolution [45,46]. By monitoring the 820 nm absorption Van Best and Mathis [45] found that following a single flash on dark adapted chloroplasts P680 is rereduced in 30 ns, probably by the secondary electron donor, Z. In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P680) is rereduced in a few microseconds in a pH sensitive reaction. Pulles et a1. [47] found that absorbance changes around 300 nm in OZ-evolving chloroplasts oscillate with flash number. One component of the oscillation has periodicity of two. It is obtained in a pure state in tris-washed chloroplasts supplied with artificial donors and it reflects the alternate one-electron reduction and oxidation of the secondary PS-II acceptor QB [47,48]. In untreated chloroplasts, however, the overall periodicity is four, which implies a contribution by donor side reactions [47,48]. Recently, Renger and Weiss [49], using trypsinized chloroplasts, eliminated the binary oscillation due to the reducing side of PS-II and observed a 320 nm absorption change which oscillated with a periodicity of four in phase with the oxygen yield as a function of the -21- number of the flash in the train. The transient 320 nm absorption change was attributed to a redox reaction within the water-splitting enzyme system (OEC). The secondary donor Z, probably a plastoquinone, is also a candidate for the 320 nm absorption change. Further investigation is necessary in order to unravel the chemical nature of the precursor. 2. Prompt Fluorescence Fluorescence is emitted by photosynthetic systems upon absorption of light. The fluorescence intensity is at any moment proportional to the concentration of excited molecules of chlorophyll. The proportionality constant, however, is time dependent. Duysens and Sweers [50] showed that the fluorescence yield of the antenna chlorophyll depended on the oxidation state of the primary acceptor QA' Oxidized QA is associa- ted with 1ow chlorophyll fluorescence whereas reduced QA results in an increase of fluorescence. In other words QA acts as if it is a "quencher" of fluorescence. To explain the low fluorescence yield observed for the P680QA' 6800A and PzaoQA' basis of experiments at 77 K, proposed that P680 is P Butler et a1., [51], on the also a quencher. Immediately after a flash, when the system is in the P2800; state, the fluorescence yield is low. It will subsequently rise upon rereduction of + . . + - . P680 by Z g1v1ng Z PGBOQA' a state w1th no quenchers. The kinetics of the rise in fluorescence are, therefore, -22- generally interpreted as reflecting the rate of reduction of P280' Fluorescence transients exhibit several phases that were extensively studied [52,53]. Sonneveld et a1. [54] have used chlorophyll a fluorescence as a monitor of + 680' dark adapted chloroplasts, they find a 35 ns reduction nanosecond reduction of P For the first flash in time. For the succeeding four flashes, the decay time of P680 is longer than 35 ns but correlated to the S states. Under steady state conditions, after damping of the period four oscillations, they find a 400 ns reduction time. 3. Luminescence Luminescence is primarily a PS-II phenomenon. In addition to absorption of light, excitons are generated by a process which may occur in the dark. The resulting afterglow or delayed luminescence (d.l.) has been studied from submicroseconds to several minutes following illumination. The spectral emission of d.1. is nearly identical to that observed for fluorescence with the maximum intensity centered around 685 nm [55,56]. This suggests that d.1. and prompt fluorescence originate, in part, from the same pigment pool. D.l. is generally believed to result from a reversal of normal photoinduced electron transport [56]. Thus, based upon the recombina- tion hypothesis, the immediate precursor of d.1. should + be the state [P680QAl’ Zankel [57] found that the yield of luminescence, measured at 90 and 500 us in a sequence of -23- flashes, oscillated in phase with 02 yields. Luminescence emissions at longer times also reflect the state of the Oz-evolving system, although in this case the yields are not in phase with 02 evolution [58,59]. An interesting modification to the delayed light experiment is a technique called electrophotoluminescence (EPL) pioneered by Arnold and A221 [60,61]. In this technique a laboratory generated electric field is imposed after illumination and a dramatic enhancement in the yield of delayed light is observed, presumably because the field has forced recombination of the photoinduced hole and electron. This technique has been used to show that delayed light and EPL are both PS-II phenomena, by demonstrating a flash number dependence on the yield of period four [62]. 4 . NMR Wydryzynski et a1. [63] have used NMR to measure the spin lattice relaxation rates (R1==1/T1) of water protons under a variety of experimental conditions and have interpreted the results as arising from photoinduced oxidation state changes of membrane bound manganese. However, Robinson et al. [64] have challenged this interpretation by showing that the kinetics of the increase in R1, produced by addition of NHZOH to thylakoid suspensions from which non-functional contamina- ting Mn(II) has been removed [65], correlate closely with the inactivation of water oxidation as monitored by assays -24- of oxygen evolution. The species producing enhanced relaxivity has a dispersion profile characteristic of Mn(II) bound to sites of reorientationally restricted mobility [65]. These results indicate that water protons do not exchange quickly (ms time scale) with protons associated with functional manganese. 5. EPR A series of articles from Sauer's laboratory demon- strated that one of the key intermediates at the donor side of PS-II can be monitored by EPR as a rapid kinetic component of the so-called "Signal II" [18,66]. Signal IIf, attributed to the oxidized form of the secondary donor Z, was first detected in tris-washed chloroplasts and its kinetic behavior was established by Babcock and Sauer [66]. Z+ was also detected in untreated, oxygen-evolving chloro- plasts and it proved to be transiently oxidized during the S2 to S3 in reference 67 assumed a single secondary donor model and the S3 to S0 transitions [STL The authors and proposed the following scheme for the reduction of Z? by the various S states: + Z- +So—+Z+Sl tkgloo us (1.4) + z- +Sl——+z+s2 tkgloo us (1.5) + z- +sz—+z +83 1:15 3400 us (1.6) + zo+s3——+z+s4 tkgl ms (1.7) -25- + .— S4-+2H20——+SO-+4H -+4e '[02 t%:<106; b) pokeweed TSF-IIa particles (0.69 mg Chl/ml), instru- ment gain==0.8><106; c) Chlamydomonas PSII particles (0.53 mg Chl/ml), instrument gain==l.25><106; d) ChZamydomonas PSII DEAE particles (0.44 mg Chl/ml), instrument gain==1><106. A modulation amplitude of 4 G and a time constant of 0.2 s were used in recording all spectra. (From ref. 92). {8 -m e- -N {8-- ton- wdmo Scot-ooze 25 8 memo-8.020 cosmos .. mrEo moo-toe .. oEoP-Egcufi -33- .:oHumuucmocoo :Hmm HH HmcmHm CH mmmmuocH woosvcH unoHH msoscHucoo 02% .mGOHumummmum umucoo coHuome HHmm onu 2H omom Hum mHszmouoHno mv can mmHoHuumm HHmm mcH>Ho>m No mnu CH omom mom mHHwnmouoHno 0mm .mumMHQouoHno pmumcoHuomumcs :H omom Mom mHstmouoHno oov UCHESmmm >Q moms muo3 mCOHumnmmmum msoHHm> one cH omom mom mchm HH Hmcun Hmuou can uanH .xumc mo mounEHummo .coHumcHEdHHH msoscHucoo >n twosocH chm HH HmcmHm mom mHHSBQOHOHEU .ooHme xumo .m mcH3oHHom chm HH HmcmHm Mom mHHmnmouoH-UAN o . . . mmHoHnumd memo em H mm o mm o mm mm mcxosofimEonb m ~m.o No.0 m.o me omH eoflumumdmudummHoHnumd aerosomeoNxb H hw.o mv.o mm.o em mHH GOHumummmumimmHoHunmm motosopmso~xb . . . mHH mma mH H mm o m o ms om omosmxom mmHoHuumm mm.o e mm.o e osw HHmm somefidm mcH>Ho>m mo . . . mummHmouoHno Ho m we H mm o omm gov omnmmsimHuu . . a mumMHmouoH 0 mo H p mo H p mm+.mmm ocH>Ho>w MO . omen omen omen -uanHc HHm -xuee- HHm eoHumummmum -Hmuou- HHm -u:mHH- HHm Mexumo- HHm A~20 Neano .Imm .mmm ecum- eoHumuHueeso eHdm -HHm- HH HaemHmIIIH mqmge -34- In the experiments reported here, we have studied the kinetic behavior of Z+ in the various preparations. The decay of Signal IIf, which follows pseudo-first order kinetics in the presence of several commonly used electron donors, was studied in the presence of various cations. Addition of cations accelerated the reduction of Z+ by negatively charged donors (e.g. ascorbate, ferricyanide) apparently due to a membrane surface charge effect. All cations used, except magnesium, had no effect on the decay time of Z+ in the presence of neutral donors. Magnesium increased the decay time of Z+ in the presence of neutral donors. At this point the effect of magnesium is not well understood. B. Materials and Methods Market spinach was used for preparing chloroplasts. Leaves were kept dark at 4°C prior to use, then washed in distilled water and deveined under low light conditions. They were broken in a Waring blender for 12 s in a standard reaction solution containing 0.4 M sucrose, 10 mM NaCl and 50 mM HEPES buffer (SHN) , pH adjusted to 7.6. The homogenate was then strained through 8 layers of cheesecloth and centrifuged for 90 s (5000 r.p.m., SS-34 Sorvall rotor) at about 4°C. For tris-inactivation the pellets were resuspen- ded in 0.8 M Tris buffer (pH==8.0) and 0.5 mM EDTA and incubated under room light, at 4°C, for 20 min. The Tris washed samples were recentrifuged and resuspended in a -35- minimal volume of SHN. Chlorophyll concentrations ranged between 4 and 6 mg chlorophyll per m1 as determined by the Sun and Sauer method [94]. 20 mg/ml spinach ferre- doxin and 5><10-4 M NADP, obtained from Sigma, were added to the final chloroplast suspension as an electron acceptor system. Photosystem II particles were isolated from mutant F54-14 of Chlamydomonas reinhardtii by using the digitonin and Triton X-100 solubilization procedure outlined in [95]; these are referred to in the text as "Chlamydomonas particles". Further fractionation of these particles to yield "Chlamydomonas DEAE particles" is also described in [95]. Triton-fractionated subchloroplast fragments (TSF-IIa particles) enriched in the reaction center com- ponents of PSII were isolated either from spinach or from pokeweed [96,97]. The above PSII preparations were kindly provided by Dr. Diner (Chlamydomonas particles) and Dr. Ke (TSF-IIa particles). EPR spectroscopy was carried out on a Bruker ERZOOD spectrometer operated at X-band. A Varian TM110 mode cavity (model E238) was fitted to the microwave bridge by using appropriately modified waveguide and impedence matching circuitry. The sample cell was a Scanlon EPR flat cell. The microwave power was 20 mW and an effective modulation amplitude of 4 G, as determined by the method of Poole [98], was used in all experiments. Because -36- of the relatively low concentration of the paramagnetic species in the chloroplast suspension (~10 uM) the time course for the transient rise and decay of Signal II was f enhanced through signal averaging in a Nicolet 1180 computer which is interfaced to the spectrometer. The instrument modifications necessary to permit efficient kinetic operation and the xenon flash lamp circuitry are described in detail in [99]. The chloroplast suspensions were kept in the dark on ice. Prior to each experiment, an aliquot of the sample was mixed with a known concentration of exogenous donors or salt. After stirring the sample with a Vortex the flat cell was filled under low light conditions. The EPR sample cell was then centered in the cavity and the detector diode allowed to stabilize. The number of scans averaged, time constant and other experimental conditions are noted in the figure captions. C. Results By using a potassium ferricyanide/ferrocyanide redox buffer system and magnesium chloride to shield surface charges, it is possible to carry out competitive flash signal averaging experiments on the transient kinetics of Signal II in the various particle preparations. Figure 6 shows kinetic traces at the indicated field values relative to the Signal II spectrum in Chlamydomonas DEAE particles. The amplitude and sense of the signal change and the -37- Figure 6. Signal II kinetic transients as a function of magnetic field in Chlamydomonas DEAE PSII particles. The spectrum of Signal II recorded in the dark is shown in (a); field positions at which the kinetic traces shown in (b) were recorded are indicated as points A, B and C on the spectrum. Field point D is the zero crossing of Signal II. For the spectrum, the following instrument parameters were used: 0.2 s time constant, 200 s sweep, 4 G modulation amplitude, 20 mW microwave power. For the kinetic traces, 200 flashes, given at a rate of 0.25 Hz, were averaged, the instrument time constant was 500 us. DEAE particles containing 0.44 mg Chl/ml were used. -33- CHLAMYDOMONAS DEAE PS II PARTICLES a) Dark Spectrum b) Kinetic Traces A B C t——I IOO ms -39- decay kinetics all indicate that the light-induced changes observed arise from Signal II. Signal shape analysis of the light induced change in spinach TSF-IIa particles yielded results similar to those of Fig. 6 for the Chlamydomonas DEAE particles. A number of other experiments were carried out in order to determine the characteristics of Signal II transient kinetics in the reaction center preparations. The results (not shown) can be summarized as follow: (1) The risetime of the signal is instrument limited at 98 us, consistent with the recent determination of the Signal IIf risetime in tris-washed chloroplasts [74] and with the properties of D1 in Chlamydomonas particles [100]. (2) The light-induced signal increase shows microwave power saturation in the 20 mW range. This behavior is characteristic of Signal II when the enhanced relaxation induced by a nearby paramagnet, presumably manganese [19] has been removed. (3) The flash-induced increase in Signal II saturates at incident light intensities which are comparable to those required to saturate Signal IIf in tris-washed chloroplasts at comparable P680 concentrations. This indicates that Signal II responds with high quantum efficiency in the particle preparations. (4) The transient response of Signal II is fairly stable in the particle preparations. We noticed, for example, decreases of 25% and 50% of the initial flash amplitude after 600 flashes on Chlamydomonas DEAE particles and TSF-IIa particles, respectively. -40- In tris-washed chloroplasts, benzidine (Em ==0.55 V), 7 is an effective donor to Signal II [36]. The data of f Fig. 7 demonstrate that in PSII reaction center preparations this species is also an efficient Signal II reductant. In Fig. 7a, the decay of Signal II in Chlamydomonas DEAE particles is at least biphasic and shows an overall half time of ~160 ms when only the potassium ferricyanide/ferro- cyanide buffer is used. The kinetic trace in Fig. 7b shows that upon addition of 40 pH benzidine the decay of Signal II is markedly accelerated; the halftime is 23 ms. In Fig. 7c the benzidine concentration dependence of the acceleration in Signal II decay is summarized. From the slope, a second order rate constant of 7x105 M'-1 s-1 can be obtained. The behavior of benzidine as a reductant to Signal II is observed in the other PSII reaction center preparations. Kinetic traces showing the effect of benzidine at various concentration levels in Chlamydomonas particles and in spinach and pokeweed TSF-IIa particles are shown in Fig. 8; the decay halftimes are given in the figure caption. The second order rate constants which we determined from the data of Figs. 7 and 8 are collected in Table II. In general, the benzidine rate constant is similar for the various particles and close to that observed for benzidine in tris-washed spinach chloroplasts [36]. The increased efficiency of benzidine in the Chlamydomonas particles was reproducible and thus appears to be a real effect. The origin of this -41- FIGURE 7 . Effect of donor addition on Signal II decay in ChZamydomonas DEAE PSII particles. The decay of Signal II in DEAE particles (0.44 mg Chl/ml) without (a) and with (b) the addition of 40 uM BZ is shown. Each trace is the average of 200 flashes, given at a frequency of 0.25 Hz. Instru- ment conditions: 0.5 ms time constant, 4 G modulation amplitude, 20 mW microwave power, field set at field point A (see Fig. 6). In (c), the BZ concentration depen- dence of the observed decay rate constant is summarized. -42- O? EE-Ne ON 0 B 2183 apnulduuv IDU5[S -43- FIGURE 8 Signal II kinetic transients in different PSII reaction center preparations. In (a) and (b), Chlamydomonas particles (0.39 mg Chl/ml) were used. No further additions were made in (a), overall decay halftime==36 ms (see text); in (b) 40 uM B2 was added as an electron donor, decay halftime==5 ms. Each trace is the average of 200 flashes, the instrument time constant was 200 us. In (c) spinach TSF-IIa particles (0.78 mg Chl/ml) were used and 40 uM BZ was added as an electron donor. The decay halftime was 30 ms, the instrument time constant was 500 us and 150 flashes were averaged. In (d), (e) and (f), pokeweed TSF-IIa particles (0.39 mg Chl/ml) were used. In (d) no further additions were made, decay halftime==l70 ms; in (e) 20 uM B2 was added, decay halftime=45 ms; in (f) 40 pH BZ was added, decay halftime = 22 ms. For these traces, an instrument time constant of 200 us was used and 255 flashes were averaged. In traces (a)-(f), a modulation amplitude of 4 G and microwave power of 20 mW were used. All kinetic traces were recorded at field setting A in Fig. 6. -44.. CHLAMYDOMONAS PARTICLES o)NA b)40p.M 82 II 50ms r———4 I50 ms I SPINACH TSF -IICJ c) 40/1M 82 200 ms POKEWEED TSF -]Io d) NA e) ZOIUJVI BZ f)4O 11M 82 #92745 I929? Isms -45- TABLE II———PSII Kinetic Parameters - Chloroplasts and Particles. . -l -1 Preparation Reagent (M s spinach chloroplasts- 5 tris washed at BZ 1' 3 x 10 spinach TSF-IIa BZ 6><105 pokeweed TSF-IIA BZ 8><105 Chlamydomonas particles BZ 3><106 Chlamydomonas DEAE BZ 7x106 part1cles spinach chl oplasts- 6 tris-washedfig ANTZP 2 7><10 Chlamydomonas particles ANTZp 6><106 Chlamydomonas particles TPB 9><105 Chlamydomonas DEAE ANT2p l.3><106 particles a From reference 36. bFrom reference 101. ~46- phenomenon is unclear although it may represent an increased concentration of the lipophilic benzidine species in the vicinity of Z in this preparation relative to the others rather than some more subtle kinetic effect. An interesting aspect of the data of Figs. 7 and 8 is that the decay halftimes of the transient Signal II increase in the presence of only the ferricyanide/ferrocyanide redox buffer system. In the DEAE particles (Fig. 7a) this halftime is ~l60 ms whereas in the parent normal Chlamydomonas particle (Fig. 8a) the decay is more markedly biphasic and shows an initial fast decrease which accounts for roughly one-third of the total decay (separate, higher resolution experiments show that this initial rapid decrease occurs with a halftime of ~1.5 ms) followed by a slower phase with a halftime of 65 ms. Diner and Bowes [100] deduced the same type of behavior for the reduction of D1 in these two types of Chlamydomonas particles from their double flash optical experiments on P680 decay kinetics, that is, that the rereduction of D1 in the normal particles is significantly more rapid than is its rereduction in the DEAE-treated material. This observation and the results in ref. 74 support the identification of D1 with the Signal II species, Z+. We also studied the effect of ADRY reagents on the decay of Signal II in Chlamydomonas particles. In tris- treated chloroplasts ADRY reagents act as reductants to 2+ in that they accelerate the decay of Z+ in a manner -47- which is first order in the ADRY reagent [101]. Figure 9 shows the effect of 20 uM tetraphenylboron (9a) or 5 uM ANT2p (9b) on Signal II amplitude and decay kinetics in normal Chlamydomonas particles and of 5 uM ANT2p on Signal 11 in the DEAE-treated material (9c). The signal amplitude in all three cases is 85-95% of that observed in the absence of the ADRY reagent. In the normal particles, the biphasic Signal II observed in the absence of the ADRY (e.g. Fig. 8a) is converted to what appears to be a monophasic decrease in signal amplitude. For tetraphenylboron we did not carry out a concentration dependence but from Fig. 9a we estimate a second order 5 M”1 s.1 for the decay of Signal II rate constant of 8><10 in its presence. This and the second order rate constants for ANT2p in the Chlamydomonas particles are summarized in Table II. The kinetic behavior of Signal IIf in tris-treated chloroplasts has been studied extensively in the past [36]. The decay of Z+ has been reported to follow pseudo-first order kinetics in the presence of several commonly used electron donors (Ascorbate, hydroquine etc.). We have investigated the effect of various cations on the decay ofzfi' in the presence of negatively charged as well as neutral donors. As shown in Fig. 10, in the presence of negatively charged donors (2 mM ascorbate) addition of salt causes a faster decay of Signal IIf, apparently the result of the screening of the negative charge of the membrane by -48- FIGURE 9. The effect of lipophilic anions on the decay of Signal II in Chlamydomonas particles PSII and DEAE PSII particles. In (a) and (b) Chlamydomonas particles (0.44 mg Chl/ml) were used. In (a) 20 uM TPB was added (decay halftime 37 ms) and in (b) 5 uM ANT2p was added (decay halftime 23 ms). Instrument conditions: 200 us time constant, 200 scans averaged. In (c) DEAE particles (0.40 mg Chl/ml) to which 5 uM ANT2p was added were used. The decay halftime is 95 ms. Instrument conditions: 500 us time constant, 200 scans averaged. Other settings as in Fig. 8. -49- o) 20 ,uM TPB b) 5pm ANT 2p ; 50ms CIDEAE Particles + 5pm ANT 2p ’ 50 ms l-——-l -50- FIGURE 10. The effect of cations on the decay kinetics of Signal II in tris-washed chloroplasts. Ascorbate (2 mM) was used as an exogenous donor. afl'No further addition; b) 200 mM d) 50 mM CaCl KCl; 0) 50 mM MgCl and e) 50 mM SrClz. 2‘ 2’ A time constant of 1 ms was used in all the experimental traces which are each the average of 120 flashes given at a repetition rate of 0.25 Hz. The decay halftimes (t%) measured from the experimental traces are: a) 350 ms; b) 290 ms; c) 285 ms; d) 94 ms; and e) 85 ms. -51- o)Asc’ b) Asc' c )Asc‘ K‘“ Mg —> —> —> d)Asc‘ e) Asc‘ C02+ Sr 2+ 200 ms |————4 -52- the various cations [102]. In the absence of any salt the decay in the presence of 2 mM ascorbate is relatively slow (Fig. 10a, t%'~340 ms), it becomes a little faster upon addition of 200 mM KCl (10b, tkav280 ms) and much faster upon addition of a divalent cation for example 2+ 2+ Ca (10d, tkrv94 ms) or Sr (lOe, ~86 ms). The ts exception in this experiment was Mg2+ (10c) which did not show the screening effect of the other divalent cations. We also studied the effect of cations in the presence of neutral donors and the results of such an experiment are shown in Fig. 11. When DPC is used as a reductant addition of Ca2+ (11b) or Sr2+ (11c) has no effect on the decay time of Signal IIf whereas addition of Mg2+ slows it down. The same behavior is observed when benzidine is used as an exogenous donor (lle-g). D.. Discussion The observation of Signal II in the particle prepara- tions, particularly in the biochemically well-characterized ChZamydomonas preparations, suggests that the donor Z is an integral component of the PSII reaction center complex and also confirms the identification of D the donor to 1! P680' w1th Z. The kinetic studies we have performed have been concerned with the reduction kinetics of Z. Our results can be compared with previous P680 Optical work from which some of the characteristics of D1 reduction have -53- FIGURE 11. The effect of cations on the decay kinetics of Signal IIf in tris-washed chloroplasts in the presence of neutral donors. a) 0.4 mM DPC; b) 0.4 mM DPC and 50 mM CaClZ; c) 0.4 mM DPC and 50 mM SrCl d) 0.4 mM DPC and 27 50 mM MgC12; e) 30 uM benzidine and 2 mM ascorbate; f) 30 uM benzidine, 2 mM ascorbate and 50 mM CaClz; and g) 30 uM benzidine, 2 mM ascorbate and 50 mM MgCl A 2. time constant of 1 ms was used in a-d and 0.5 ms in e-g. The experimental traces in a—d are the average of 120 flashes given at a repetition rate of 0.25 Hz, whereas traces in e-g are the average of 150 flashes given at the same rate (0.25 Hz). The decay halftimes (t8) measured from the kinetic traces are: a) 64 ms; b) 60 ms; c) 60 ms; d) 104 ms; e) 16 ms; f) 16 ms; and g) 29 ms. o) DPC e) 82 -54- b) DPC 2 .\Wii I I ZOOms r—a f) 82 r C02+ i “I IOO ms c)DPC Sr2+ g)BZ Mg2+ dIDPC Mg2+ -55- been inferred. For example, Diner and Bowes have concluded that D1 is rereduced more rapidly in their normal Chlamy- domonas particles than it is in DEAE particles [100]. The reduction kinetics of 2+ which we measure in these two types of particles are in agreement with their results (Figs. 7a, 8a, see above). A corollary conclusion which can be drawn from a comparison of the optical work on the particles [100] and our results is that the kinetic characteristics of Signal II and of the reduced acceptor Q- A are quite dissimilar. From the second order rate constant for Q; reoxidation by ferricyanide determined in ref. 100, we would predict that Q; is reoxidized in the Chlamydomonas particles under the conditions we have typically used with a halftime of less than 1 ms. Signal II, on the other hand, decays with a halftime approximately two orders of magnitude longer, indicating that in the Chlamydomonas particles 0; does not give rise to a Signal II-like spectrum. This point is important to establish in that if the iron associated with QA were removed during particle purification, we might expect a free radical type spectrum [97,103]. Our data indicate that this is not the case and are in agreement with recent PSII acceptor side EPR studies in the Chlamydomonas particles [104]. Renger and Reuter [105] have recently shown that D: reduction is accelerated both by reducing agents and ADRY reagents. The faster rereduction -56- of Z+ in the presence of these species in PSII particles support the identification of Z and D1. A study of the effect of various cations on the decay of Signal II has revealed a specific effect of magnesium on the kinetic behavior of 2+. As shown in Figs. 10 and 11, magnesium, in addition to its screening effect on the negative charge of the membrane, also causes a decrease of the decay time of Z+ in the presence of neutral or negatively charged donors. At this point, the mechanism of this effect is not well understood. It is possible that magnesium causes a conformational change of the membrane at the region of Z, which alters its kinetic properties. Further kinetic, as well as biochemical, studies of the system are needed in order to understand this specific effect of magnesium. [CHAPTER III. EXOGENOUS VERSUS ENDOGENOUS ACCEPTORS IN PHOTOSYSTEM II INHIBITED CHLOROPLASTS A. Introduction DCMU is known to block the oxidation of the reduced form of the primary acceptor QA by the secondary acceptor QB (or B) [50,106]. As a result, Q; accumulates in the light and further electron flow through Photosystem II is inhibited. In the presence of DCMU, Q; can be oxidized by addition of ferricyanide. Two different mechanisms have been proposed to account for this observation. In the first, ferricyanide is postulated to oxidize 0; directly [107,108]. Itoh [108] monitored the oxidation rate of the primary acceptor in the presence of exogenous electron acceptors by measuring the induction of chlorophyll fluorescence in the presence of DCMU. He found that the apparent rate constant for the oxidation of Q; changed widely when either the medium pH or salt concentration was varied. By using acceptors having positive, negative, or no charge, he concluded that the membrane surface potential in the vicinity of the acceptor side of PSII had a dramatic effect on the value of the rate constant -57- -58- observed for the reoxidation of Q; by charged species. The interaction of the charged artificial acceptor with Q; was explained in terms of the Gouy-Chapman theory [109,110]. In this treatment the apparent rate constant for the reoxidation reaction is dependent upon the local concentra- tion of the acceptor at the membrane surface. The surface concentration of this species is a function of its charge and of the electrical potential difference at the surface of the membrane with respect ot the bulk aqueous phase. In the case of ferricyanide, for example, the relationship between its surface concentration and the surface potential, wo, is given by the following equation: [Ferricyanidels-Ys = [Ferricyanidelb-Yb exp(3Fwo/RT) (3.1) where s and b stand for surface and bulk phase, respectively, and y denotes the activity coefficient. The rate of Q; oxidation can be given as dIle - —7fi;— = k°logllFeIIICle-ys (3.2) and de’] _ _ _7fi%_ = k[QA][FeIIICN]b (3.3) where k° is the rate constant with respect to the surface activity of ferricyanide and k is the apparent rate constant (see ref. 108). From Eqs. (3.1) and (3.3), the relation between R and k° follows as k = k°yb exp(3FwO/RT) (3.4) -59- In the second model, ferricyanide was envisioned as being the redox mediator in titrating an endogenous acceptor which is the species actually responsible for the reoxidation of 0;. From the titration experiments, a midpoint potential of ~400 mV has been postulated for this endogenous species. An identification of this acceptor with cytochrome b has been ruled out [111]. 559 In.a recent review, Bouges-Bocquet summarized the properties of this species and pr0posed that it be referred to as AH [8]. This AH seems to be the same species observed in [68,100,111-116]. Babcock and Sauer [116] reported that the DCMU inhibition of Signal IIf, which arises from the oxidized form of the P680 donor, 2? [74,921, was relieved if sufficiently high reduction potentials (Eh >480 mV) were maintained in tris-washed chloroplast suspensions. The explanation they offered for their results was that by keeping the potential high they maintained A (in Bouges- H Bocquet's notation [8]) in its oxidized form so that, under their flashing light conditions, it could act as an additional acceptor for Q; in their DCMU-inhibited chloroplasts. In the work reported here we have used EPR spectro- scopy to reinvestigate the conditions for Signal IIf formation in tris-treated, DCMU-inhibited chloroplasts. We are able to interpret our data without invoking an A H species and thus we conclude that the reoxidation of Q; -60- in DCMU-inhibited preparations depends on the effectiveness of ferricyanide in interacting directly with QA' B. Materials and Methods Chloroplasts were prepared from market spinach and tris-washing and chlorophyll determinations were carried out as described in Chapter II. The chlorophyll concentra- tion in the EPR experiments was 3 mg/ml. The buffer system used consisted of 0.4 g sucrose, 0.05 g HEPES and 0.01 3 NaCl which was adjusted to pH 7.6. Reagents were of the highest purity commercially available and were used without further treatment. Redox potentials of the chloroplast suspensions were measured with a platinum /Ag-AgCl electrode (Broadley-James Corp.) which was connected to an Orion 701 A pH/mV meter. The electrode was initially calibrated vs. saturated quinhydrone [117] and was periodically checked by using an equimolar mixture of ferri- and ferrocyanide in the above buffer (SHN). The potential for the couple was measured to be 430 mV in the absence of and 458 mV in the presence of 40 mg Mg++. The redox potentials measured in specific experiments are noted in the figure captions. Electron paramagnetic resonance (EPR) spectroscopy was carried out by using a Bruker 200D instrument operating at X-band and interfaced to a Nicolet 1180 computer. Instrument modifications and the flash lamp are described in [99]. Light induced changes in Signal IIf amplitude were measured by setting the magnetic -61- field value at the low field maximum of the Signal II spectrum; a modulation amplitude of 4 G was used. The flash repetition rate, instrument time constant, and number of passes averaged in specific experiments are detailed in the figure captions. C. Results Upon inhibition of the oxygen-evolving complex, elec- tron flow to Z? is interrupted, its lifetime is increased, and its EPR signal is referred to as Signal IIf. In a signal-averaged, flashing light experiment, the formation of Z? is inhibited by DCMU [100]. Previously [101] we showed that the DCMU inhibition of Signal IIf formation is relieved if relatively high concentrations of FeIIICN and divalent cations were added. Ferricyanide addition restores Signal IIf amplitude under these conditions by regenerating the photoactive state, Z P680 QA' from Z P680 Q; in the dark time between flashes. The action of FeIII CN can be explained by either of the two models described above. To decide between the alternatives, we have explored the ferricyanide effect in more detail by examining its dependence on dark time between flashes, salt concentration, redox potential and acceptor ionic charge. As shown in Fig. 12a, the amplitude of Signal IIf is a function of the dark time (td) between successive flashes in DCMU inhibited, tris-washed chloroplasts. At -62- FIGURE 12A. Effect of the dark time (td) between flashes on the magnitude of Signal IIf [A==(AIIf)/(A$;:)' where AIIf max is the amplitude in is the measured amplitude and AIIf the absence of DCMU]. 3 mg Fe(CN)2-, 20 mg Mg++ and 100 uM DCMU were added to a suspension of tris-washed chloroplasts at pH 7.6. The instrument time constant was 1 ms; 150 flashes were averaged. The dashed lines show the theoretical fit of these data according to Eq. (3.8). Signal II f Magnitude .0 or -63- [.0- A. 3mM FemCN, ZOmM Mgclz IO -54- FIGURE 12B . Graphical determination of the apparent rate constant of Q; reoxidation by ferricyanide (see text for details). ~65- td(sec) -66- sufficiently long dark times the effect saturates and full Signal II amplitude, as determined by controls carried f out in the absence of DCMU, is observed. The data in Fig. 12A were obtained with 3 mg FeIIICN and 20 mg MgCl2 and show half maximal Signal IIf amplitude at tdEEZ sec. The interplay between Signal IIf amplitude and flash repetition rate in the presence of DCMU is influenced by both the salt and the ferricyanide concentration. As shown in Fig. 13, the oxidation of QA at constant FeIIICN concentration and dark time between flashes is facilitated by addition of Mg++, apparently due to the effect of the divalent cation on the surface potential in the vicinity of System II as reported by Itoh [108]. The Mg++ effect of Fig. 13 is nonspecific since it is also given by other 4. +, Sr++) as well as monovalent (K+, Na ) divalent (Ca+ cations, with divalents being more effective (not shown). III Figure 14 shows the effect of Fe CN concentration on the amplitude of Signal IIf under conditions where Mg++ was present and the ratio of ferri- to ferrocyanide was kept constant. Increasing the ferricyanide concentration in the absence of ferrocyanide gave similar results, i.e., an increase in Signal IIf magnitude with increased acceptor concentration (data not shown). With ferricyanide alone, however, we found fluctuations in redox potential; by maintaining a constant ferricyanide to ferrocyanide ratio, the redox potential remained constant at ~460 mV during a given experiment and did not change significantly between -67- Figure 13. Effect of Mg++ concentration (MgClz) on the magnitude of Signal IIf. 3 mg Fe(CN)2- and 100 uM DCMU were added to a suspension of tris-washed chloroplasts at pH 7.6. The instrument time constant was 1 ms; 150 flashes were averaged at a frequency of 0.33 Hz (td = 3 sec). -68- $5 385: om d nywmwmmu o . h 20.3mm 26m v.0 0.. annuubow ’11 mums -69- FIGURE 14 . Effect of ferricyanide concentration on the magnitude of Signal IIf. The amounts of ferri- and ferrocyanide shown above were added to a suspension of tris-washed chloroplasts at pH 7.6, which contained 40 mg Mg++ and 100 uM DCMU. The redox potential in all samples was'v460 mV. 150 flashes were averaged at a frequency of 0.5 Hz and with an instrument time constant of 1 ms. -70- Eat Edam: .. 2er v N 4&0 - j - u - 06mm . 3 Aces. .2506 . — P n - b to O apruyubow J11 Ioufils 0.. -71- samples. The increase in IIf magnitude with increasing ferricyanide concentration apparent in Fig. 14 can be interpreted according to the direct acceptor model for ferricyanide action: according to Eq. (3.1) the surface concentration of ferricyanide is increased when its bulk concentration is increased, which, because of Eq. (3.2), results in a faster reoxidation of QR. The results above indicate that, at constant redox potential (i.e. constant ferricyanide to ferrocyanide concentration ratio), the amplitude of Signal IIf observed depends on the flash repetition rate, the absolute ferricyanide concentration and the concentration of the shielding cation. If we maintain these factors constant and decrease the redox potential of a chloroplast sample by adding additional ferrocyanide, we observe a progressive decrease in the Signal IIf amplitude when divalent cations are used to screen the membrane surface charge, but not when monovalent cations are used as the shielding species. Typical results from such a set of experiments are shown + in Fig. 15. Using just FeIIICN (6 ml!) and Mg+ (40 mg), full Signal II amplitude is generated (Fig. 15a). Upon f addition of FeIICN (60 mg), the redox potential of the chloroplast suspension decreases to 405 mV and a partial inhibition in Signal IIf formation is observed (Fig. 15b) which is relieved when we increase the dark time between flashes (Fig. 15c). If the same experiments are carried -72- FIGURE 15 . Effect of negatively charged species on Signal IIf in tris-treated DCMU-inhibited chloroplasts, pH 7.6, in the presence oflu)m§ Mg++ and 6 mg Fe(CN)2-. a) No further addition, td=3.5 sec; b) 60 mg Fe(CN)g-, td=3.5 sec; c) 60 mI_VI_ Fe(CN)2-: t :8 sec; d) 200 mg SO (NaZSO4), d 4 td==3.5 sec and e) 200 mg_so:, td==8 sec. The redox potential in (a) was 582 mV, in (b) and (c) 405 mV and in (d) and (e) 566 mV. The instrument time constant was 1 ms and each trace is the average of 150 flashes. -73- b)FenCN c)FeICN o) td=3.5 sec id = Bsec fl“- F ‘ 1 d) 30: e) so: 1d=3.55ec [d = 85ec M —> —> 200 ms |——4 -74- out with 500 mg KCl (or NaCl) as the only screening species, we find maximal Signal II amplitude independent f of the ferrocyanide concentration (not shown). Two explanations for the observations in Fig. 15a and 15b are possible as follows. 1) In an A endogenous H acceptor model, the added ferrocyanide lowers the solution redox potential sufficiently to reduce a significant fraction of AH in the dark. As a result, only partial reoxidation of Q; occurs on subsequent flashes and formation of Z? is suppressed. 2) In an exogenous acceptor model, by introducing high concentration of FeIICN we decrease the apparent rate constant of the reaction between Q; and FeIIICN and under these conditions an increase in the dark time (td) is necessary for complete reoxidation of Q; to occur between flashes. That full Signal IIf amplitude can be generated even in the presence of high FeII CN concentrations simply by increasing the dark time between flashes (Fig. 15c), or by using monovalent instead of divalent cations to screen the surface charge, appears to favor the second explanation. High concentra- tion of FeIICN could decrease the rate of the reoxidation reaction simply by decreasing the surface concentration of FeIIICN. The decrease of the surface concentration of FeIIICN could be either the result of a simple displacement III II of Fe CN by Fe CN (according to Stern's theory there is a limited number of sites at the membrane [118]) or the result of a decrease in the surface potential (i.e., the _75_ surface potential becomes more negative) caused by addition of the polyvalent anion, FeIICN (see below). To distinguish between the exogenous and endogenous acceptor models we used high concentrations of NaZSO4 instead of FeIICN at constant FeIIICN concentration. IICN in that it is The sulfate anion is simlar to Fe polyvalent. It is, however, redox inactive and therefore its addition does not significantly decrease the solution redox potential (see caption to Fig. 15). The results of this experiment (Fig. 15d) show that a decrease in Signal IIf amplitude occurs with increases in so: concen- tration. Moreover, the sulfate induced suppression of Signal IIf amplitude, like that observed in the presence of ferrocyanide, could be relieved by increasing the dark time between flashes (Fig. 15e). (Controls carried out 2SO4 had no effect on the extent or kinetics of Signal IIf formation in the absence of DCMU showed that 200 mM Na and decay when td==3 sec was used.) These experiments indicate that the decrease in Signal IIf amplitude observed upon increasing the FeIICN concentration (Fig. 15b) is not the result of a decrease in the solution redox potential but rather arises from a perturbation to the surface potential induced by this polyvalent anion. As a result of the altered surface potential, the surface IIICN is decreased. concentration of Fe We noted above that one mechanism by which the poly- valent anion induced inhibition of Signal IIf amplitude -76- could occur was by simple displacement of ferricyanide in the double layer. However, the inhibition occurred only IICN, 30:) were added to solutions 2 when polyvalent anions (Fe in which divalent cations (e.g. Mg +) were used to screen membrane surface charge. No inhibition in Signal IIf amplitude occurred with either monovalent anions (e.g., C1-) or with monovalent screening cations (K+ or Na+). These observations suggest that ion-pair association occurs between the added polyvalent anion and the divalent which results in a decrease in the effective concentration of the screening cation [119]. Such an ion pair association will be more pronounced when a divalent cation is used to shield the surface charge for two reasons. First, the higher charge of the divalent compared to the monovalent leads to more efficient ion-pair formation [119]. Second, the divalent cation is effective in screening surface charge at much lower concentrations compared to that of the monovalent and, because of its lower concentration, its screening capacity is more susceptible to the effects of ion-pair formation. A decrease in the effective concentration of the divalent cation results in an incomplete shielding of the membrane surface and, consequently, a decrease in the surface concentration of FeIIICN. As predicted by this model, the FeIICN inhibition observed in Fig. 15b was relieved by addition of higher concentrations of MgCl2 (data not shown). -77- The results above indicate that, under our experimental conditions, Q; acceptor rather than by an intermediate endogenous species is reoxidized directly by the exogenous and that the factor which controls the oxidation of Q; is not a relatively high redox potential in the medium but rather the presence of an efficient acceptor. Since the midpoint potential of the ferricyanide-ferrocyanide couple was measured to be 458 mV (in the presence of 40 mM Mg++), it was difficult to decrease the solution potential to values much below 390 mV and, at the same time, keep the concentration of ferricyanide at levels where it would be an effective acceptor. Thus, we looked for an acceptor with a lower midpoint potential. Itoh [108] has shown that the positively charged species, PMS, is a very efficient acceptor under conditions where the surface potential of the membrane was negative. Figure 16a shows that in the presence of PMS, and under conditions where A should be reduced (Eh==260 mV, measured before H and after the experiment), 0; time between flashes. This result strongly suggests that is reoxidized in the dark PMS reacts directly with QR. The decay halftime of Signal IIf in the presence of PMS is fast owing to the reduction of Z? by the reduced form of PMS at that potential. When the potential was increased to 520 mV IIICN, the decay rate of Z? was slowed by addition of Fe down because the concentration of reduced PMS was minimized (Fig. 16b). We also tried to use various quinones to -73- FIGURE 16. Phenazinemethosulfate (PMS) as an artificial acceptor in tris-treated DCMU-inhibited chloroplasts at pH 7.6. a) 1 mM PMS; redox potential 260 mV; b) 1 mM PMS and 1 mM Fe(CN)g-; redox potential 520 mV. 150 flashes were averaged at a frequency of 0.25 Hz. The instrument time constant was 200 us in (a) and 1 ms in (b). -79- o)PMS b)PMS Fem CN 40 ms -30- adjust the redox potential, but our kinetic data were complicated by the presence of very strong, g==2.0 EPR signals from the semiquinones. PMS also shows a weak EPR signal in this region (g‘~2.0), but it does not effect the kinetic traces of Signal IIf, as shown by repeating the experiment at various magnetic field positions on the Signal II spectrum. D. Discussion In the presence of DCMU, oxidation of Q; by the endogenous secondary electron acceptor is blocked. The EPR results presented above indicate that the release of this DCMU inhibition observed upon ferricyanide addition results from a direct oxidation of Q; by the added acceptor. It thus becomes unnecessary to postulate an endogenous component, A to explain this observation. To develop H’ this in more detail, we begin with Itoh's observation that after continuous illumination the oxidation of Q; in the presence of ferricyanide in DCMU inhibited chloro- plasts occurs by a pseudo-first order reaction with respect to the bulk concentration of ferricyanide [120]. From Eq. (3.3) we can calculate the amount of QA which remains in the reduced form [le after time, t, under a t! given set of experimental conditions as III - _ - -k [Fe CN] °t where [Q-] A 0 is the concentration of Q; produced by the ~81- flash. From Eq. (3.5) we calculate the halftime of the reaction to be t3: = 11“; (3.6) k[Fe CN] b From Eqs. (3.5) and (3.6), and assuming that single- turnover, saturating flashes are used, we obtain [Q 1 /[Q l = 1-2‘t/t% (3 7) A t A ° where [QA]t is the amount of QA in the oxidized form after time t and [QA] is the total concentration of the acceptor irrespective of its valence state. Thus, in a series of flashes td time apart the amount of QA oxidized between two flashes will be given by Eq. (3.7) with t==td. Because the amount of Signal IIf generated by a flash under steady state conditions is proportional to the amount of QA oxidized between flashes we can write max _ _ -td/tg AIIf/AIIf — 1 2 (3.8) or l-A = z-td/tk (3.9) where A = A /Amax This can be rewritten as IIf IIf‘ III log (l-A) = -o.43 k[Fe CNlbtd (3.10) Application of Eq. (3.10) to the data of Fig. 12a gives a plot shown in Fig. 12b. The slope of the straight line III which results is -0.16 and therefore, -k[Fe CN] -82- TABLE III———Apparent Rate Constants:flmrQ; Oxidation by Ferricyanide in the Presence of Various Concentrations of MgClz. m9“) (mM) 10"2 xk (114—1 s’l) 0 0.3 10 0.9 20 1.2 (0.43) = -0.16. Since the concentration of FeIIICN used is 3 mM_we calculated the apparent rate constant k to be 2 M 5"1 under the experimental conditions of Fig.12. 1.2 XlO Using the data of Fig. 13 and Eq. (3.10), we can calculate the value of the apparent rate constant for various concentrations of Mg++. The results are summarized above in Table III. From Itoh's work (ref. 108, Fig. 2) we obtain a k value of 0.4XlO2 M-1 s.1 for the reoxidation of Q; by FeIII CN in the presence of 20 mM_MgC12, which is in good agreement with our k value for similar conditions. Therefore, the results from two different kinds of experiments, Itoh's work on fluoresence detection of Q; reoxidation [108] and our present data on Z? by EPR detection, converge and indicate that the ferricyanide relieves DCMU inhibition by oxidizing Q; directly. -33- According to the model proposed here, the decrease in Signal IIf observed by Babcock and Sauer [116] when they decreased the solution potential by titrating the ferricyanide containing suspension with ascorbate, can be attributed to the decrease in the ferricyanide concentration rather than to the drop in the redox potential upon addition of ascorbate. Although these authors were aware of possible ferricyanide specific effects and repeated their titration with a different redox mediator, cyanotungstate, it is likely that the surface charge effects apparent for ferricyanide will also be in force for cyanotungstate. From the results and arguments presented above, it is apparently unnecessary to invoke the high potential acceptor component, A in order to rationalize steady H' state EPR results in tris-washed chloroplasts. The AH species has also been postulated to account for results obtained in low temperature optical and EPR experiments and in room temperature fluorescence measurements (see [8]). These studies involved essentially single flash techniques applied to dark-adapted material. They are thus quite distinct in terms of experimental protocol from our measurements and it becomes uncertain as to whether an extrapolation of our results to these systems is appropriate. Nonetheless, it is possible to explain these results in terms of direct exogenous acceptor oxidation of 0; rather than in terms of the endogenous —84- AH species. Two additional observations regarding these earlier studies support this hypothesis: a) the FeIIICN/FeIICN redox couple has been used to poise the solution redox potential in all previous studies in which AH has been proposed [68,100,111-116] and b) no known redox component occurs endogenously in PSII which has the AH midpoint potential (since A [111] ) . is not cyt b H 559 CHAPTER IV. THE ROLE OF ADRY REAGENTS IN DESTABILIZING HIGH-POTENTIAL OXIDIZING EQUIVALENTS IN CHLOROPLAST PHOTOSYSTEM II A. Introduction At least three components have been implicated in the molecular events which lead to oxygen evolution in chloro- plast Photosystem II (for reviews, see [8] and [121]). The first of these, the reaction center chlorophyll complex P680' is involved in photon absorption and subsequent primary charge separation. Good progress in understanding these processes has been made recently owing to the strong analogy which has emerged between the photochemistry which occurs in the Photosystem II and that which occurs in the photosynthetic bacterial reaction center [97,122, 123], although the state of chlorophyll aggregation in the two different reaction centers may be distinct [72]. The second component, usually designated as D or as Z, l is an intermediate electron carrier which appears to be a quinone species [81,85,116,124]. When oxidized during P680 rereduction, Z? gives rise to EPR Signal IIvf in oxygen evolving chloroplasts [125] and to EPR Signal IIf in inhibited chloroplasts [91]. The kinetics of the -85- -86- electron transfer reactions of this intermediate have been studied extensively by EPR [36,67,90,102], by optical [45,126,127], by luminescence [128] and by fluorescence [54] techniques and a reasonably clear picture of its involvement in P680 rereduction has emerged. The possibili- ty that a parallel donor alternates with Z as a P680 reductant has also been raised [8]. The third component on the oxidizing side of Photosystem II is the oxygen evolving complex (OEC) which carries out the actual water splitting reaction. Kok and coworkers have shown that the oxidizing equivalents required in this process are accumulated linearly with P680 turnover and have developed the S-state terminology to describe the formal oxidation state of the oxygen evolving complex [14]. Proton release data suggest that the higher oxidation states may actually correspond to partially oxidized water intermediates which remain bound to the OEC [22,23,29,l30]. Manganese has been implicated as a cofactor in the water splitting reaction (for review, see [131]); recent work has provided additional support and insight into the role of manganese in this process [19,20,31]. In the reactions which occur on the oxidizing side of PSII, these components (P Z and OEC) are often 680’ postulated to form a linear chain so that oxidizing equivalents generated by photon absorption in the reaction center are transferred via Z into the CBC. Within this formulation the redox potential of these components can be -37- roughly estimated. At pH 7, the midpoint potential of the water/oxygen couple is 815 mV; however, photosynthetic water oxidation appears to occur such that the protons released in this process appear in the acidic internal thylakoid volume [22] where the steady state pH value may be as low as 4.5. Thus the OEC operates under condi- tions where the midpoint potential of its reductant, water, is at least 965 mV. From work which has been done with both P680 [45,126] and with EPR Signal IIV [67], it f appears that the equilibrium constants for the reaction of P680 with Z and of Zt with the OEC must be at least 10. Thus the midpoint potential of Z? is more positive by at + least 60 mV relative to that of the OEC and that of P680 is at least 60 mV more positive than that of the Z/Zt couple. Despite these strongly oxidizing potentials, the water oxidizing system appears to be remarkably specific for H20. Commonly used reductants are completely ineffec- tive in competing with water for the oxidants generated in PSII [132]. Only those species which are structurally similar to H20 (e.g. H202, NHZOH) appear to be oxidized by PSII at the expense of water. Part of this specificity, no doubt, arises from the extremely rapid redox reactions which occur during the transfer of photogenerated oxidizing equivalents from P680 through Z to the OEC. These reactions proceed with halftimes in the sub-ms time range and even the slowest step in this process, the actual generation of -88- 02, occurs with a 1 ms halftime [67,133]. Thus exogenous reductants, which react with Z? in tris-washed chloroplasts with second-order rate constants in the 102-107 M-1 5-1 range [36], are excluded at normal concentration levels (5100 uM) simply by the kinetics of the membrane bound transfers. The intermediate states of the OEC, however, are reasonably stable, even though they are held at high redox potentials, and only decay (i.e. are reduced to lower formal oxidation states) when the interval between successive absorbed photons extends well into the seconds range [13,14]. The chemical basis for the remarkable stability of the higher S states is at present unknown. Over the past decade, a class of compounds has been found which increases the rate by which the intermediate formal oxidation states of the OEC decay [134-137]. Much of this work has been carried out by Renger and coworkers who refer to these species as ADRY reagents. Little is known regarding the mechanism by which they destabilize higher S-states. However, some members of the ADRY class are remarkably efficient at S-state deactivation even at very low concentrations (~1 per PSII reaction center), and, as a consequence, models in which ADRY reagents activate a cyclic pathway for the reduction of the OEC, rather than participate directly in the redox chemistry, appear to be favored. That one of the requirements for the ADRY effect appears to be an acidic proton on a lipophilic anion has also attracted comment [138]. Recently, Renger and -39- Eckert [139] and Renger and Reuter [105], using optical techniques to monitor P680 decay, have shown that in tris-washed chloroplasts, ADRY reagents promote the rereduction of Z? following flash excitation. Thus ADRY reagents appear to be general in increasing the rate at which the highly oxidizing equivalents generated by PSII are reduced. In the work described here, we have used EPR spectroscopy to monitor the effect of ADRY reagents on the behavior of Z? in tris-washed chloroplasts in signal-averaged, flashing light experiments. Our results can be interpreted to indicate that ADRY reagents act by reducing Z? directly in a bimolecular reaction with second- 5 7 M-1 s-l. order rate constants on the order of 10 -10 B. Materials and Methods Chloroplasts were prepared from market spinach and tris-washing and chlorophyll determinations were carried out as described in Chapter II. Chlorophyll concentrations in EPR experiments ranged from 3 to 6 mg/ml. The buffer system used in most experiments consisted of 0.4 M sucrose, 0.05 M HEPES, 0.01 M NaCl which was adjusted to pH 7.6. Addition of other reagents is indicated in the figure captions as is the buffer system used when experi- ments were carried out at pH values other than 7.6. Ferredoxin and NADP were added as an acceptor system [36] to maintain electron flow under signal-averaging conditions. Standard chemicals were obtained commercially and were of -90- the highest purity available. DCMU was from Dupont and was recrystallized prior to use. ANT 2p was a gift from Dr. G. Renger; ANT 2a was a gift from Dr. R. Blankenship. Care was taken to maintain the organic solvent volume at less than 1% the volume of the chloro- plast suspension when they were used in order to solubilize the various reagents. At this concentration the solvents used had no discernible effect on the reactions being monitored. Electron paramagnetic resonance (EPR) spectroscopy was carried out with a Bruker ER 200D instrument operating at X-band and interfaced to a Nicolet 1180 computer. The instrument has been modified slightly primarily in that we increased the effective Q of the phase sensitive signal detector by imposing either a passive 50 KHz high pass filter or a PAR 189 selective amplifier between it and the detector diode preamplifier. The rationale for and effects of these modifications have been discussed by Yerkes [99]. A Varian TM mode cavity (E238), modified for operation with the Bruker spectrometer [l9], and Scanlon TM mode flat cell (S-8l4) were used in the experi- ments described. In flashing light experiments, a critically damped xenon flashlamp (ILC, Sunnyvale, CA), which provides saturating pulses 14 us in duration (full width, 1/3 maximum intensity), was used in conjunction with laboratory constructed capacitor discharge and timing circuitry [99]. Light induced changes in Signal IIf -91- amplitude were measured by setting the magnetic field value at the low field maximum of the Signal II spectrum; a modulation amplitude of 4 G was used. The flash repetition rate, instrument time constant and number of passes averaged in specific experiments are detailed in the figure captions. C. Results In oxygen-evolving chloroplasts, Z? is rereduced by the OEC in the submillisecond range following excitation [67,125]; the free radical state of Z observed under these conditions is referred to as Signal IIvf’ Upon inhibition of the OEC electron flow to Z? is interrupted and the lifetime of the free radical is extended well into the ms time range. Under these conditions, the Z? free radical is referred to as Signal IIf and its reduction occurs at the expense of endogenous donors (e.g. ascorbate) in the chloroplast suspension [91]. Upon addition of exogenous donors, the lifetime of Z? is decreased owing to a direct bimolecular reaction of the donor with Zt. This pathway is the principal route by which exogenous donors restore PSII electron flow in inhibited chloroplasts [66]. The efficiency of various donors in restoring this flow can be judged by the second-order rate constant for their reaction with Z? which have been tabulated recently [36]. Figure 17 shows typical Signal IIf data we obtain with the hydroquinone/ascorbate couple. In the absence of -92- FIGURE 17 . The effect of donor addition on the decay of Signal IIf in tris-washed chloroplasts. In (a) 2 mM sodium ascorbate was added as a donor, the observed rate constant for Signal IIf decay is 4.2 5.1; in (b) 100 uM hydroquinone and 2 mM sodium ascorbate comprised the donor system, kobs==l6.4 5.1; in (c) 200 uM hydroquinone and 2 mM - -l - bs-—34 s . The instrument sodium ascorbate were used, ko time constant in (a) was 1 ms which was decreased to 500 us in (b) and (c). Each trace is the average of 180 flashes given at a frequency of 0.25 Hz. -93- ors- loom-e D T we 00%. 01-21093 (341-0 -94- exogenous donors the decay halftime is slow, typically in the 500 ms-—l s time range [91]. Addition of ascorbate at substrate levels increases the decay rate (Fig. 17a) and increasing concentrations of the more efficient donor, hydroquinone, accelerates the Z? rereduction further (Figs. 17b,c). From these data, k2, the second-order rate constant for the reaction between HZQ and Z? (Eqn. 4.1) + k2 + HzQ-+Z°-———+Z-+H2Q- (4.1) can be extracted by plotting the observed (pseudo-first order) rate constant (kobs) versus the H2O concentration corresponding to the particular value of k0 The value bs' we obtain in the series of experiments of Fig. 17, l.7><105 M-1 3.1, is in good agreement with the value reported earlier [36]. If we now carry out a similar experiment but replace the exogenous donor couple with an ADRY reagent we can probe the mechanism by which this class of compounds accelerates the reduction of 2?. Figure 18 shows typical data we have obtained with the ADRY reagent, ANT 2p. In the absence of the ADRY reagent, Signal II exhibits f a slow decay similar to that described in conjunction with Fig. 17. Increasing ANT 2p concentration from 2.5 uM (Fig. 18a) through 5 uM (18b) to 15 uM (18c) leads to progressively more rapid Signal II decay. Figure 18d f shows a first-order plot for the decay of Signal IIf in -95- FIGURE 18. The effect of ANT 2p on the decay kinetics of Signal IIf in tris-washed chloroplasts. ANT 2p at the indicated concentrations was used in (a), (b) and (c). A time constant of 500 us was used in all three experimental traces which are each the average of 180 flashes given at a repetition rate of 0.25 Hz. In (d), a semilog plot of the decay of Signal IIf in (b) is presented. A hand- drawn curve was fitted to the data and the amplitude of Signal IIf at various times was extracted from this curve. -96- 0 d)5,u.M J l J o 20 4o 60 1(ms) -97- the presence of 5 uM ANT 2p. Its linearity indicates that the time course of Z? reduction is exponential and hence that the reaction is proceeding under pseudo-first order conditions. By plotting kobs for the experimental traces in Fig. 18 versus the corresponding ANT 2p concentration we can extract the second-order rate constant which describes the decay of Z? in the presence of this ADRY reagent (see Figure 20 and Table IV). We have investigated a number of other ADRY reagents including picrate [140], CCCP and ANT 2a, in their effects on the decay kinetics of Zr. The data for picrate are shown in Fig. 19 where we show Signal IIf kinetic transients for picrate concentrations between 15 uM and 180 uM (Figs. l9a-e). The increase in Signal II f decay rate is apparent as the halftime of exponential TABLE IV-——Calcu1ated Second Order Rate Constants for the Rereduction of Z? in the Presence of Various ADRY Reagents. ADRY Reagent kail-l s-l) ANT 2p 2.7 x lo6 cccp 1.9 x lo6 ANT 2a 0.5 x lo6 picrate 0.2><106 -98- FIGURE 19. The effect of picrate addition on the decay kinetics of Signal IIf in tris-washed chloroplasts. In (a)-(e) 9 mM K3Fe(CN)6 and 40 mM CaCl2 were added to a suspension of tris-washed chloroplasts at pH 7.6 and picrate at the indicated concentrations was added. Note that the time axis is compressed by a factor of 2 in (a) compared to the other traces in the figure. In trace (f), the K3Fe(CN)6 and CaCl2 were omitted from the reaction mixture which was 180 uM in picrate; in (g) 9 mM K3Fe(CN)6 and 180 uM picrate were present but the CaCl2 was omitted. The instrument time constant was 500 us; 150 flashes were averaged at a frequency of 0.25 Hz. 0)l5,u.M 200 ms 1f“ e)|80,u. M lOOms -99- Picrate b)30,u.M GOFM d)90p. M c) lOO ms M f lOOms 1 f)|80p. M (3)80}; M -FeC -C02 -C0 lOOms IOO ms -100- FIGURE 20. Graphical determination of the second-order rate constants for the decay of Signal IIf in the presence of various ADRY reagents. The observed rate constant for the decay of Signal IIf in the presence of the indicated ADRY reagent is plotted against the corresponding ADRY concentration. The slope of the straight lines which results is the second-order rate constant. These are tabulated in Table IV. -101- Om. 26.-oi a 8.24 . 88 4 amaze. 0 gene x 7&2- oo_ 4 q -102- decay of Signal IIf decreases progressively from 126 ms in Fig. 19a to 16 ms in Fig. l9e. The picrate experiments were carried out in the presence of 9 mM ferricyanide and 40 mM Ca2+ owing to an apparent inhibiting effect on the reducing side of PSII by this ADRY reagent, similar to that described for other ADRY reagents [141], which leads to an inhibition of Signal IIf formation at high picrate concentrations (Fig. 19f). Addition of ferri- cyanide alone relieves this inhibition to some extent, (Fig. 19g), but full development of IIf requires both salt addition and ferricyanide. The salt requirement is non- specific and most likely involves a surface charge shielding effect as originally described by Itoh [142]. The kinetic data we have obtained for four ADRY reagents are collected in Fig. 20 where we have plotted the observed first—order rate constants (kobs) for Signal IIf decay versus the corresponding ADRY concentra— tion. That these plots are linear for all four reagents indicates that the relationship, k S==k[ADRY], holds. ob From the slopes of the lines we can extract values for the second-order rate constant, k, for each of the ADRY reagents. These values are summarized in Table IV and indicate that the effectiveness of ADRY reagents in stimulating 2* reduction in tris-washed chloroplasts increases in the order picrate‘k2. For the decay of Signal II which reflects fl . . + the reduction time course of Z-, we have -105- + + k2[Z-][Dr]-+k3[Z-ADRY][Dr] (4.9) To obtain the observed first-order dependence on the ADRY reagent concentration (figs. 18-20) we must postulate that the equilibrium in reaction (4.6) is established quickly, i.e. that k1, k_1:8>k2 or R3. Then we solve the equilibrium expression in (4.6) and express [ZTADRY] in terms of [2?] and [ADRY] as 32L -1 [ZTADRY] = [ADRyllzT] = KIADRyllzT] (4.10) W and substituting into (4.9) we obtain dlzT] _ I - dt — kobslz ] (4.11) where kobs = (kzlnr]-+k3K[Dr][ADRY]) (4.12) In the absence of an ADRY only the first term in Eq. (4.12) survives, whereas in the presence of an ADRY reagent the second term dominates. In this formulation the second-order constants for the various ADRY reagents in Table IV correspond to the product k3KlDr]. The donor Dr may be a soluble component, either endogenous (e.g. ascorbate [93]) or exogenous, or it may be a membrane bound component. In the experiments described below we have attempted to test these two models in order to decide which is more appropriate. -106- If Dr is a soluble component, then the two models above make different predictions as to the decay of Signal IIf under conditions where both an ADRY reagent and an added donor are present. In the direct reduction model, Z? may be reduced either by the ADRY or by the added donor and thus we expect the observed rate constant to be simply the sum of the rate constants observed when each of the components is present separately. The cataly- tic model, on the other hand, predicts that an ADRY reagent should sharply increase the rate of Z? reduction when present with added donor owing to the increased concentration of the faster reacting ZTADRY conformation. Thus the catalytic model predicts that there should be a synergistic effect when exogenous donor and an ADRY reagent are added to the same chloroplast suspension. Figure 21 presents the results of two sets of experiments which were carried out to test the contrasting predictions. In Fig. Zla-c CCCP was used in conjunction with the HZQ/ascorbate donor system. In the presence of 5 uM CCCP alone, the decay halftime was 75 ms (Fig. 21a); with 150 uM HZQ alone, the decay halftime was 20 ms (Fig. 21b). When the ADRY and the exogenous system were used together, the decay halftime was 21 ms (Fig. 21c). These results indicate that there is essentially no synergism between the two compounds in the rate at which Z? is reduced. In the second series of experiments (Figs. 21d-h), ANT 2p was used in conjunction with the HzQ/ascorbate —107- FIGURE 21 . The effect of an ADRY reagent in conjunction with an electron donor system on the decay kinetics of Signal IIf. In (a)-(c) the ADRY, CCCP, and the donor system, hydroquinone/ascorbate, were used. The CCCP concentra- tion in (a) and in (c) was 5 uM; no CCCP was present in (b). Hydroquinone (150 uM) and sodium ascorbate (2 mM) were added in (b) and (c), but were absent in (a). A time constant of 1 ms was used for traces (a)-(c), each of which is the average of 150 flashes given at a repeti- tion rate of 0.25 Hz. In traces (d)-(h) the ADRY, ANT 2p, and the hydroquinone/ascorbate donor system were used. In (d) and in (f), the hydroquinone concentration was 20 uM and ascorbate was present at 2 mM; the donor system was omitted in the other traces. The ANT 2p concentration in traces (e)-(h) was 10 uM; it was omitted from the chloroplast suspension used in obtaining trace (d). In (g), 12 mM K3Fe(CN)6 and 40 mM CaCl2 were present and in (h) 40 mM CaCl2 was added. An instrument time constant of 500 us was used in obtaining the experimental data in (d)-(h). 180 flashes, given at a rate of 0.25 Hz, were averaged to obtain the final decay curves. -108- o)CCCP b) HQ 0) CCCP \M r HQ [ [ IOO ms [ A? d)HQ e)AN12‘,p f)ANsz HQ . MM 4 i 200 ms g)ANT2p h)ANT2p FeCN 2+ 002‘ MM 200 ms[ -109- couple. The conditions were chosen so that Signal IIf decay in the presence of only H2O (20 uM, tg==l90 ms, Fig. 21d) was slower than that in the presence of ANT 2p alone (10 uM, t%:=38 ms, Fig. 21e). When the two were used together (Fig. 21f), the observed halftime, 38 ms, again indicates that there is no synergism between an ADRY reagent and a soluble electron donor. Figure 21g is a control carried out to demonstrate that the decrease in Signal IIf amplitude in Figs. 21e and f relative to that in 21d is the result of a slight PSII reducing side inhibition induced by ANT 2p, analogous to that observed above for picrate, which can be relieved by addition of ferricyanide and a divalent cation. Figure 21h shows that the acceleration in Z? decay rate observed in Fig. 21g comes about because of the presence of the divalent cation, most likely because at the pH used in the experi- ment ANT 2p is largely in its anion form [135] and subject to surface charge effects as has been shown previously for the ascorbate monoanion [36,102]. The results of Fig. 21 indicate that if the catalytic role envisioned for ADRY reagents in the second model described above is correct, the source of electrons for the reduction of Z? cannot be a soluble reductant. A second potential reductant pool which may be activated by ADRY addition involves reduced components in the electron transport chain on the acceptor side of PSII [105]. The results of experiments designed to test this ~110- hypothesis are shown in Fig. 22. When DCMU is added to tris-washed chloroplasts, Signal IIf formation and decay are no longer observed under signal-averaged, flashing light conditions [116]. This inhibition can be relieved by addition of ferricyanide and a divalent cation to insure approach of the oxidant to the thylakoid membrane [102,142] (Fig. 22a, decay halftime==85 ms). Upon addition of the ADRY, ANT 2p, a stimulation in the rate of Z? reduction is observed (Fig. 22b, t% = 15 ms). This result apparently eliminates B, the PQ pool and cytochrome b559 as reductants susceptible to ADRY action owing to the facts that DCMU inhibits transfer of electrons to B and PO and that in the presence of ferricyanide we expect these species as well as b559 to remain oxidized in the dark. The oxidizing conditions were apparent in that high concentra- tions of the P;00 free radical were present in these samples. Figure 22c is a control which shows that the rate of Z? reduction is independent of the presence or absence of DCMU. Figure 22d shows that if the divalent cation and ferricyanide are omitted, no Signal IIf formation in the presence of DCMU is observed. This experiment indicates that ADRY reagents are unable to catalyze a direct ZfQ- back recombination which agrees with a similar conclusion obtained earlier by Renger et al. [136]. We conclude from the experiments of Fig. 22 that ADRY reagents can exert their accelerating -111- FIGURE 22. The effect which conditions on the acceptor side of Photosystem II exert on the decay kinetics of Signal IIf in the presence of an ADRY reagent. The decay of Signal IIf was recorded in tris-washed chloroplasts in which the final concentration of added reactants was as follows: (a) 100 uM DCMU, 12 mM K Fe(CN)6, 40 mM 3 CaClz; (b) 100 HM DCMU, 12 mM K3Fe(CN)6, 40 mM CaCl 2' 10 0M ANT 2P; (C) 12 mM K3Fe(CN)6, 40 mM CaClz, 10 uM ANT 2P; (d) 100 uM DCMU, 10 pM ANT 2p. For each trace, 180 flashes were averaged at a flash repetition rate of 0.25 Hz. The instrument time constant was 1 ms. -112- neoc- s-f- .- .- - am too :8 20.....- :8 .524 28..- anpzq zed“- 2283 354-0 3283 328-0 -1l3- effect on Z? decay independent of the redox state of the acceptor side components and thus the primary model of ADRY action does not appear to involve catalysis of cyclic flow to Z? from these compounds. Taken together, the results of Figs. 18-22 provide no evidence for the catalytic model of ADRY action, rather they indicate that these reagents are likely to reduce Z? in tris- washed chloroplasts in a bimolecular reaction as outlined in the direct reduction model. Renger has pointed out that effective ADRY reagents possess an acidic proton which, upon dissociation, generates a lipophilic anion. Moreover, the anion form appears to be more effective in stimulating ADRY action than is the neutral form [143]. The results of Fig. 23 Show that similar behavior is exhibited by ANT 2a (pKa==7.0, [134]) during its reaction with 2?. At pH 5.8, where this species is over 90% in its protonated form, the decay halftime of Z? is 53 ms (Fig. 23a); at pH 8.2, where 90% is in the anion form, the decay halftime of Signal IIf decreases to 33 ms (Fig. 23b). Assuming pseudo-first order behavior and the pKa value above, one calculates that the anion form of ANT 2a is 1.7 times . . . + . more effective than the neutral speCIes in z- reduction. D. Discussion In addition to their uncoupling activity [144] and inhibitory action on the reducing side of PSII [141], ADRY -114- FIGURE 23. The effect of pH on the decay of Signal IIf in tris- washed chloroplasts in the presence of ANT 2a. In (a) tris-washed chloroplasts were resuspended in a buffer which contained 0.4 M sucrose, 0.01 M NaCl and 0.05 M MES. The pH was 5.8 and 15 uM ANT 2a was present. In (b) a buffer consisting of 0.4 M sucrose, 0.01 M NaCl and 0.05 M HEPES at pH 8.2 was used. The ANT 2a concentration was 15 uM. Each trace is the average of 180 flashes, 0.25 Hz repetition rate and 1 ms time constant. -115- ANTZCJ b)pH=8.2 -> 70 ms’ -116- reagents destabilize oxidizing equivalents generated by P680' States and species which are susceptible to this destabilization effect include the OEC intermediates 52 and S3 [105,136], the free radical species Signal IIS [145-147] and the electron transport component Zr [139, this work]. Moreover, ADRY reagents alter significantly the behavior of two other species which appear to be linked to PSII function, cytochrome b559 and carotenoids [77,148-151]. In the work reported here, we have shown that the rate of Z? decay shows a first-order dependence on the concentra- tion of ADRY reagent and have listed in Table IV the second-order rate constants for this reaction for four ADRY reagents: ANT 2p, CCCP, ANT 2a and picrate. These data show that the efficiency of these four species in exerting the destabilization effect varies by a factor of about 14 and decreases in the order ANT 2p2>CCCPI>ANT 2a > picrate. This ordering and the magnitude of the second order rate constants provide a basis from which the relationship between the effect we observed for ADRY reagents in their interaction with Z? and that observed for their destabilization of S2 and S can be explored. 3 Renger, Vater and their coworkers have reported S2 and S3 decay curves for a variety of ADRY reagents at various concentrations [105,135-137,139,140,l43]. In general the decay of the OEC intermediates in the presence of an ADRY follows an exponential time course (although there are -117- exceptions [140]) and the rate constant for this process increases as the ADRY concentration is increased. This behavior is qualitatively similar to that which we observe for Z? decay in the presence of an ADRY and implies that $2 and S3 exhibit pseudo-first-order decay behavior upon ADRY addition. To put this observation in more quantitative terms, we have tabulated data on the ADRY-induced destabilization of OEC intermediates which have been reported over the past decade (Table V). The halftimes (tg) were taken from the data reported and converted to pseudo-first-order rate constants by using the relationship, kobs = [BIZ/tk. The apparent second- order constants, k, were calculated as kobS/[ADRY]. The results show that there is remarkably consistent behavior observed for the various species and that the second- order formulation appears reasonable. For ANT 2p, for example, the second-order rate constant varies by no more than a factor of two even when the concentration is varied over an order of magnitude. Moreover, inspection of the ANT 2s and ANT 2a data indicates that this factor of two may be more a function of experimental error (e.g. two separate measurements at l><10-6 M ANT 2a shows a twofold variation), than a real concentration dependence in the second-order rate constants. Focusing on the data in Table V for the four ADRY reagents we have studied in their interaction with 2?, we see that the efficiency of these species declines in the order .mmscmucou > magma -118- meme-om n mmcnmao m3 e3xa.m m.m 36 73:. mm 92¢ 23 s3 x «.3 5m 35 73 x 4 mm 92..- s3 e3 x a mg 35 73 x N mm aza- mom x m n mmosmac m3 03 x 35 To m3 m..3 x 3 «am 92¢ 23 e3 x 5 03 3.0 ea3 x 3 an 924 m3 m3 x as m.m 36 73 x m 9.. 92¢ G3 @331- m.m 3m;- 73x a 3. 92¢ m3 m3 x m -..N 3.0 73 x m 3 92¢ m3 m3 x s TN 35 73 x m 3 92¢ m3 h3.1.13 m3 :5 73.3 am 924 . . and a. E- mmm AHIm alz-u- -Hlm- x Am- u newumuucwocoo ucmmmmm .memEou mcH>Ho>MIcmm>xo mcu mo mmumum coflumpflxo Hmsuom stmwmmfiucoflum>fiuommo on» Ca mucmmmmm wand msofluw> How museumcou wumm “mono pcoomm pmamHsonolll> mqméa .ucmumcoo mumu umpno pcoomm on» NO wsam> mmmum>m may mcflcHEumump ca boondocw no: -_oma.moa- muasmwn msofiuomm o» pma w>mn awe >uw>wuom xocmu mufl wumc3 mponuomam Escflumam mnmn m um use pmwuumu .1 -119- 23 «.3 x m.m mm .o m 73 accession-33c IN 23 m3 x m.m mm;- 33 73 mush-3d 23 m3 x Rs 5.5 8.0 73 mooo s3 A.13 x m6 m6 :5 73 noon mQNVAw.~ n wmcxmau m3 m3x33 ~.~ 35 73.. N 3 92¢ 23 G3 x s5 5o o3 73 mm 92..- m3 m3 x 33 GA 3.0 73 mm 92..- m3 m3xm3 m5 NA 73.; mm ezg who r E- .mmm fun 7:- v- $12 6- -n- u soflmfismosoo usmmmmm .Umscflucoo > magma -120- ANT 2p >ANT 2a2>CCCP2>picrate. The relative inefficiency of CCCP with respect to ANT 2a in Table V may be an example of experimental variation in that in ref. 135, Renger indicates the reverse, i.e., that CCCP is more efficient than ANT 2a. In addition, the ADRY efficiencies, as judged by the value of the second-order rate constants, vary by a factor of about 16 as one goes through the series of four. Finally, the magnitude of the rate constants for a given ADRY reagent in Tables IV and V are within a factor of four (e.g., Table IV: 6 1 -l k(ANT 2p) =2.7 x10 13" s ; Table v: k(ANT 2p) = 9 x106 {1 s'l). These observations, in conjunction with the data in Fig. 23 indicating that the anion is the more effective form, suggest that the mechanism by which ADRY reagents destabilize $2 and $3 in oxygen-evolving chloroplasts is the same as that which we observe for the ADRY-induced rereduction of Z? in tris-washed chloroplasts. Note, however, that we do not intend to convey the idea that ADRY reagents destabilize S2 and S3 by intercepting oxidizing equivalents at 21- in water-splitting chloro- plasts. The data of Table V indicate that S2 and S3 destabilization is observed in the hundreds of ms time range under the ADRY concentration conditions usually employed, whereas it has been shown [67,125] that Z? transfers its oxidizing equivalent into the OEC in the sub-ms range in 02 evolving chloroplasts. Thus in -121- uninhibited chloroplasts the site of ADRY action is the OEC itself, whereas in tris-washed chloroplasts the much longer lifetime of 27 makes it susceptible to ADRY attack. Within the context of the data of Tables IV and V, it is worthwhile to consider briefly the effect of ADRY rea- gents on the decay of Signal IIS. Lozier and Butler [145] originally demonstrated that a number of reagents, including CCCP, destabilized the free radical species which gives rise to this EPR signal so that its decay halftime decreased from hours in the absence of CCCP to seconds in the presence of this reagent. They also showed that subsequent illumina- tion led to Signal IIs regeneration [145]. Subsequently, Babcock and Sauer confirmed these results and showed that the Signal IIS precursor, which they designated as F, was oxidized to its free radical form by a redox interaction with the OEC intermediate states S and 8 [93,146]. They 2 3 also carried out a detailed study of the CCCP concentration dependence of the Signal IIS decay rate and presented data on the decay halftime of Signal IIS which showed that increasing CCCP levels led to increased destabilization of the radical (see Fig. 3 in ref. 146). From the decay halftime data they presented, we have calculated observed rate constants and have plotted kobs versus the CCCP concentration in Fig. 24. The four points fall on a reasonably good straight line and from the slope we obtain an apparent second order rate constant for the -122- FIGURE 24. Graphical determination of the second-order rate constant for the decay of Signal IIS in oxygen-evolving chloroplasts in the presence of CCCP. The data from Fig. 3 in ref. 146 were used to obtain the pseudo-first order rate constant, kobs' for the decay of Signal IIs at various CCCP concentrations. This rate constant is plotted against the corresponding CCCP concentration in order to determine the second-order rate constant as the slope of the straight line which results. -123- O- goo-Anode- m m e m o 1 q q u d J u q ( |_S)€O| XSQOX -124- 2 1 CCCP-induced destabilization of Signal IIS of 6.5><10 M- s-l. This value is approximately three orders of magnitude smaller than the second order rate constant for the CCCP induced destabilization of Z? (Table IV) or for the destabilization of 82 and 83 (Table V). This observation indicates that, although IIS is mechanistically similar to Z? and to S2 and S3 in its susceptibility to the action of ADRY reagents, it is much more sluggish in terms of its response time. Nonetheless, these results show that the action of ADRY reagents appears to be general in that oxidized species generated by the light reaction of PSII are susceptible to destabilization by ADRY reagents and that the decay process which results shows a first order dependence on ADRY concentration. The data we have presented above can be used to provide several insights into the mechanism by which ADRY reagents act on oxidizing equivalents generated by PSII. Previous considerations of this problem [105,135- 140] had focused on essentially two alternative possibili- ties as follows: (1) ADRY reagents are redox active cofactors and act by reducing the stored oxidizing equivalents directly; (2) ADRY reagents act in a catalytic manner and, by inducing conformational or configurational changes in PSII, facilitate a cyclic reaction which accelerates the decay of the stored oxidizing equivalents. While several ADRY reagents are clearly redox active (e.g. -125- tetraphenylboron [77,152] and dichlorophenol-indophenol [140]), the catalytic mechanism has generally been preferred on the basis of stoichiometric arguments over the direct reduction mechanism [e.g. 137]. Consistent with the catalytic mechanism is the well-known observation that cytochrome b559 is photooxidized, apparently by PSII, in the presence of ADRY reagents [148,151] and recent discussion of the ADRY catalytic mechanism has focused on an involvement of b in the process [77,105,139]. 559 In the experiments presented above we have attempted to explore these two possibilities. The first-order dependence on ADRY reagent concentration which is observed for Z? decay in tris-washed chloroplasts (Fig. 20) appears to be characteristic of the general mechanism by which these species destabilize PSII oxidizing equivalents (see Table V and Fig. 24) and leads one to the alternative kinetic models, direct reduction (Equations 4.2-4.5) versus catalytic (Equations 4.6- 4.12) presented and discussed briefly in the Results section. In developing experimental tests aimed at identifying the source of electrons for the catalytic mechanism we have been able to eliminate an ADRY-catalyzed increase in the efficiency with which soluble endogenous or exogenous donors reduce 21 (Fig. 21). Likewise, the data of Fig. 22 argue against the possibility that the source of reducing equivalents mobilized by ADRY reagents is either on the reducing side of PSII or originates in -126- cytochrome b559. In fact, the decay of Z? in the presence of an ADRY reagent is remarkably insensitive to any perturbation supplied to the acceptor side of the photo- reaction. For example, if one compares the decay of Z? in the presence of 10 uM ANT 2p under conditions where electron transfer is blocked at Q (Fig. 22b) to the analogous experiment in which there is no acceptor side inhibition (Figs. 22c,Zlg), one finds that the flash- induced Z? amplitude and decay time (14 ms) remain constant. Similarly, if one carries out the experiment in the absence (Fig. 21b) or presence of (Fig. 22c,Zlg) ferricyanide, it is again apparent that the decay of Signal IIf in the presence of 10 uM ANT 2p remains constant at 14 ms. The amplitude difference in these experiments is the result of the slight acceptor side inhibition of PSII observed by us and others [14] at relatively high ADRY concentrations. Finally, if one takes into account the salt effect described below, the data of Figs. 21e,f and g indicate that the reduction of Z? by ANT 2p proceeds independent of the redox state of cytochrome b559. These observations eliminate both this species and PSII acceptor side components as necessary sources of reducing equivalents in an ADRY-catalyzed Z? rereduction reaction. The only significant means by which we have been able to influence the decay rate of 27 at a given ADRY -127- concentration is through salt addition. For example, in Fig. 21e, Z? decays with a 38 ms halftime in the absence of salt which decreases to the 14 ms lifetime of Fig. 21h when 40 mM Ca2+ is added. This salt is independent of the identity of the divalent cation and is the sort of screening effect one expects for an anion, in this case the deprotonated form of ANT 2p, interacting with a reaction partner associated with a negatively charged membrane [142]. It has been shown recently that the inner membrane surface in the vicinity of PSII is negatively charged and that this surface charge does influence the reduction kinetics of Z? by anionic species [102]. The results discussed above argue against the catalytic model of ADRY action, but are clearly consistent with the predictions of the direct reduction mechanism. The latter model, however, requires a substrate level reductant (Dr in Equation 4.3) in order to regenerate the reduced form of the ADRY reagent from its oxidation product. That this requirement is significant is evident from the sustained ADRY effect observed even at low reagent concentration which was originally noted and commented on by Renger [137] and confirmed in the experiments reported here. An inspection of the literature indicates that this reductant pool is likely to consist of membrane components, such as carotenoids, chlorophyll and protein, which are susceptible to non-specific -128- oxidation by ADRY(ox). This possibility is indicated by the results of Yamashita et al. [149] and of Itoh et al. [150] which showed that addition of the ADRY, CCCP, to either oxygen-evolving or tris-washed chloroplasts led to the photobleaching of both carotenoids and chlorophylls. Similarly, Homan has noted photodestruction of PSII in the presence of the ADRY reagents, CCCP or tetraphenylboron, and has suggested that these species funnel oxidizing equivalents generated within the photosystem into destructive side reactions [152,153]. Finally, it is 559 is available, it is oxidized in the presence of an ADRY well established that if reduced cytochrome b reagent and light by a Photosystem II-driven reaction [77,148,151]. Taken together, these results suggest that a fairly non-specific oxidation reaction occurs upon illumination of ADRY—treated chloroplasts and that the direct reduction model outlined above may be rewritten as the following two step sequence: 21’ +ADRY—L-rz +ADRY(ox) (4.13) carotenoid carotenoid+ chlorophyll fast chlorophyll ADRY(ox)4- b2+ -———-+ADRY(red)4- b3+ 559 559 soluble soluble oxidation reductants products (4.14) Although we cannot provide definitive evidence to eliminate a catalytic mechanism which invokes an ADRY- -129- mediated direct oxidation of carotenoids and chlorophylls by 2?, we favor the mechanism outlined in Equations 4.13 and 4.14 for several reasons. First, it accounts for the exponential decay of Signal IIf we observed in the presence of low concentrations of an ADRY reagent under an extended flash regime (typically 150-200 flashes) without postulating a specialized structural arrangement between 27 and carotenoid, chlorophyll or b Second, this 559' model accounts for the oxidation of b in the presence 559 of an ADRY reagent if the cytochrome is in its reduced state prior to initiation of a flash train. However, it does not make the oxidation of this species mandatory for ADRY action, consistent with our data described above and with experimental results which indicate that in the presence of FCCP, b559 oxidation is non-stoichiometric [A.R. Crofts, personal communication]. Third, it explains the inhibition of carotenoid bleaching which is observed if an exogenous reductant is added to CCCP treated chloroplasts [149,150]. Fourth, it accommodates those ADRY reagents which are known to be redox active (e.g. tetraphenylboron, dichlorophenolindophenol). Finally, the model is supported by Renger and Reuter's observation of redox activity for ANT 2s at a platinum electrode [105] and by our own observation of redox activity for several ADRY reagents (picrate, ANT 2p, ANT 2a, CCCP) by cyclic voltammetry (Ghanotakis,:LEl and Babcock,G.T. unpublished). Picrate has also been studied elsewhere and its electrode -130- reactions appear to be complex [154]; we are in the process of exploring the electrochemical behavior of various ADRY reagents in more detail. The scheme presented in Fig. 25 summarizes the model developed above for the behavior of ADRY reagents on the oxidizing side of PSII. The strongly oxidizing species generated by P680 are susceptible to reduction by an ADRY reagent in a manner which depends on the ADRY concentration, its second order rate constant and the lifetime of the specific intermediate. In OZ-evolving chloroplasts, ADRYs act primarily on the states S2 and S3 and, in a much slower reaction, on the species FT which gives rise to Signal 115. In chloroplasts inhibited at the OEC, the lifetime of Z? is extended significantly and it becomes a site of ADRY action at fairly low ADRY concentrations. The interaction between the ADRY reagent and the PSII oxidant generates the oxidized form of the ADRY reagent, itself a good oxidant which is able to oxidize the indicated chloroplast components in a fairly non-specific reaction. The net result of these reactions is the regeneration of the ADRY reagent, the oxidation of a variety of endogenous components and the accelerated decay of stored oxidizing equivalents in Photosystem II. -l31- FIGURE 25. A model for the role of ADRY reagents in destabilizing oxidizing equivalents generated in PSII. The solid arrows denote proposed reactions in oxygen-evolving chloroplasts, dashed arrows indicate proposed reactions in tris-washed chloroplasts and the dotted lines indicate inhibitor action. See text for other details. -132- m-co-oapmm maocmooxmy mono £38829 moose-Boo. CHAPTER V. HYDROXYLAMINE AS AN INHIBITOR BETWEEN Z AND P680 IN PHOTOSYSTEM II. A. Introduction Exposure of isolated thylakoid membranes to NHZOH is known to be a mild treatment for inactivation of the oxygen evolving reaction system [32-35]. Cheniae and Martin [33] reported that the mechanism of NHZOH inacti- vation of the oxygen evolving complex (OEC) is different from that of tris-inactivation: inhibition by tris is accelerated by low light intensities [30,31], whereas NHZOH produces a rapid inactivation at low concentrations in the dark. In the same work [33], the authors report that NHZOH-inhibition occurs by two different mechanisms. One is the extraction of Mn from the photosynthetic membrane while the other mechanism, which has an immediate but reversible effect, was not localized. Sharp and Yocum [155], using NMR relaxation techniques, proposed that higher S-states are immune to attack by NHZOH and that NHZOH-induced Mn extraction occurs only from the lower S-states. Den Haan et al. [156] studied the fluorescence kinetics in ChloreZZa Pyrenoidosa in the -133- -134- presence of NHZOH and suggested that an inhibition site could be localized between Z, the physiological donor, and the oxidized reaction center pigment, P680' This inhibition was reversed during illumination of a washed sample. These authors also reported that for a series of high frequency flashes in the presence of NHZOH, P680 was rereduced by a back reaction with the reduced form of the primary acceptor, QR. In this work we have used EPR spectroscopy to monitor the formation of Signal IIf and P680 directly in order to localize the immediate NHZOH inhibition observed in the past [33,156]. Since the EPR signal which arises from P680 is reported to have the same shape and g-value as that which arises from P700’ we used PSII preparations free of Photosystem I in addition to unfractionated thylakoids in these experiments. In agreement with [156], we find that NHZOH exerts an immediate and reversible inhibition which results in a blocking of electron flow from Z to P680' B. Materials and Methods Intact thylakoid membranes were prepared as described in Chapter II. Subchloroplast membranes, free of Photosys- tem I and capable of oxygen evolution, were prepared as in [88] except that the initial Triton extraction and subsequent washings were carried out at pH 6.0. We have found that by using lower pH values during the isolation, -135- PSII particles with higher 02 rates result. The buffer system used contained 0.4 M sucrose, 0.01 M NaCl and either 0.05 M HEPES for pH 7.6 or 0.05 MES for pH 6.0. Chloro- phyll concentrations were determined as described in [94]. EPR spectroscopy was carried out on a Bruker ER-200D spectrometer operated at X-band and interfaced to a Nicolet 1180 computer. Instrument modifications as well as the xenon flash lamp circuitry and the protocol followed in signal-averaged, flashing light kinetic experiments are described in [99]. C. Results When 02 evolution is inhibited at the oxygen evolving complex, the lifetime of the intermediate electron carrier, 2?, is extended into the hundreds of ms time range and becomes detectable as EPR Signal IIf. Yocum et a1. [19] reported that incubation of chloroplasts in the dark with NHZOH followed by washing of the system with buffer resulted in complete inhibition of 02 evolution and conversion of Signal IIvf to Signal IIf. When we incubated chlorOplasts with NHZOH in the dark and then, without removing the inhibitor, transferred them to the EPR spectrometer we found that neither Signal IIvf nor Signal IIf was present. After washing the system twice with buffer Signal IIf could be observed. We repeated the above experiment with tris-treated chloroplasts and noted the same behavior, i.e., the presence of NHZOH -136- in the system inhibited Signal IIf formation, whereas its removal allowed full development of Signal IIf. PSII subchloroplast preparations showed analogous behavior in the presence of hydroxylamine except that lower concentra- tions of NHZOH were required. Indeed when we measured the O2 rates of PSII preparations immediately following addition of NHZOH (Fig. 26), we noticed that low concentra- tions of NHZOH were very effective in inhibiting 02 evolution. Intact chloroplasts required higher concentra- tions of NHZOH in order to obtain the same extent of inhibition. For example, under the conditions of Fig. 26, 50% inhibition of 02 evolution in PSII preparations occurs at ~l mM NHZOH; in chloroplasts under these conditions ~5 mM NHZOH was required for 50% inhibition. Reflecting the behavior of 02 evolution in response to NHZOH addition, Signal IIVf of untreated PSII prepara- tions and Signal IIf of tris-treated preparations disappears and a new signal, which rises within the 50 us response of our instrument and had a decay halftime of less than 200 us, is present (Fig. 27). Removal of NHZOH from tris-treated PSII preparations restores the system to its initial state (full Signal IIf, no new signal, data not shown). The shape of the new signal, as determined by signal-averaged experiments at different field values, is shown in Fig. 28. Its peak-to-peak width is 7-8 Gauss and its g-value is 2.002; these values are in agreement -137- FIGURE 26. Effect of NHZOH concentration on the O2 preparations at pH 6.0. PSII preparations (10 ug rate of PSII Chl/ml) were suspended in the polarograph vessel with the exogenous acceptors, Fe(CN)2- (3.5 mM) and 2,5- dichloro-p-benzoquinone (250 uM). Addition of NHZOH was followed by immediate illumination to determine the rate of 02 evolution. -138- 3.5 crow-z- 0.. q O O N O 0 q. |uofiu1.q/zo sa|owT7' -139- FIGURE 27. Kinetic transient of the P680 EPR signal. 1 mM Fe(CN)2-, 1 mM Fe(CN)2— and 2 mM_NH20H were added to a suspension of PSII particles at pH 6.0. The instrument time constant was 50 us; 150 flashes were averaged at a frequency of 0.1 Hz. -140- -l41- FIGURE 28. Shape of Signal P680 (solid line) in PSII preparations obtained by kinetic experiments at the indicated field values. The shape of Signal IIS is also shown (dotted line). Insert: Saturation properties of Signal P680 in PSII preparations at pH 6.0. Each point represents the amplitude of the P680 EPR signal at the indicated microwave power. The conditions were the same as those in Fig. 27. Ema-<9 Ibwzmmhm 04m.“- -142- Oan GOV-m Ommm 80m 9me _ . a _ . D/ \\ // w-BE- «to-son 26300-2- 00 N IO / \ \ o._ . ._ om . o z \ (n O BONIubow (oubus -l43- with those reported for P680 [68,70]. Quantitation of the new signal, by double integration and correction for the instrument time constant, showed that it is stoichiometric (i20%) with Signal II The saturation f. properties of the radical are shown in the insert to Fig. 28 which indicates that it saturates at relatively high power. Compared to this radical, Signal I (EPR signal of P700) saturates at somewhat lower power which could be the result of environmental differences between the two species. D. Discussion The most common inhibitory site on the oxidizing side of Photosystem II is at the oxygen evolving complex itself (e.g. [19]) and NHZOH has a well-known inhibition at this site [33,155]. Fluorescence data indicate, however, that NHZOH also produces an inhibition in PSII which is probably localized between Z, the physiological donor, and the oxidized reaction center pigment, P680 [156]. In the work reported here, we have used EPR to show that NHZOH does block the flow of electrons from Z to P680 in a way which is completely reversible. In the absence of NHZOH, Z, which can be identified with + 680 variety of chlorOplast preparations [121], reduces P680 in a reaction with a halftime.515 us. Under these D1 [74], the immediate electron donor to P in a -l44- conditions we observe the EPR signal of 2?, produced after reaction of Z with P680’ but we are not able to follow the EPR signal of P680 because of the response time of our instrument. Upon addition of hydroxylamine, electron flow between Z and P680 is interrupted and P680 is reduced by back reaction with the reduced form of the primary acceptor, Q- This reaction is much slower A' compared to the reduction of P680 by Z [121] and we can follow it with our experimental apparatus. Thus, we can explain the NHZOH inhibition of PSII with the scheme shown in Fig. 29. According to that scheme, NHZOH has two effects. In the first, NHZOH interrupts the flow of electrons from the S-states to Z by extracting Mn [33,155] which results in the conver- sion of Signal IIvf to Signal IIf. In the second mode of inhibition, which appears to be unique to NHZOH, the flow of electrons from Z to P is interrupted 680 in a completely reversible manner. The mechanism of the second inhibition is not clear and it could be a direct effect on Z, for example formation of a Z-NHZOH complex, or an effect on the membrane environment of Z. That P680 is rereduced by Q; in the inhibited centers, however, implies that the chemical and not the redox properties of NHZOH are responsible for this inhibition. -l45- FIGURE 29. A model for the inhibitory role of NHZOH in Photosystem II. See text for details. -146- acceptors ] CHAPTER VI. INHIBITORY TREATMENTS OF OXYGEN EVOLUTION AND THEIR EFFECTS ON MANGANESE CONTENT, zt BEHAVIOR AND POLYPEPTIDE COMPOSITION. A. Introduction Signals IIvf and IIf are light-generated free radicals detected by EPR in chloroplast thylakoid membranes [67, 116]. Both signals arise from the same species, 2?, which is an intermediate electron carrier between the Photosys- tem II reaction center, P680’ and the oxygen evolving complex (OEC). Signal IIv is the EPR signal of the f radical Z? observed in Oz-evolving chloroplasts and has a very fast decay (sub-msec) as the result of its reduction by the various S-states. Signal IIf, on the other hand, is the EPR signal of 21 observed under condi- tions where oxygen evolving capacity has been inhibited by various treatments. One of the treatments used to uncouple the oxygen evolving complex from the rest of the photosynthetic chain is incubation with high concen- tration of Tris buffer [29-31]. Tris treatment releases functional manganese from the oxidizing side of Photo- system II by attacking the higher S-states, and under these conditions the electron flow from OEC to Z is -147- -l48- interrupted and the lifetime of the free radical is extended well into the ms time range. Under these conditions, the Z? free radical is referred to as Signal IIf. Upon addition of exogenous donors, the lifetime of Z? is decreased owing to a direct bimolecular reaction of the donor with Z? [36]. In addition to the change in the kinetic behavior of Z, tris-treatment also changes the power saturation properties of Signal II. Warden et al. [90] found that Signal IIvf could not be saturated at microwave powers up to 200 mW whereas Signal IIf was inhomogeneously broadened with saturation at ~25 mW. These findings were interpreted to indicate that IIVf was in the vicinity of a strongly relaxing species, probably Mn(II), associated with photosynthetic oxygen evolution. Another treatment which releases functional manganese from OEC is incubation with higher concentrations (~5 mM) of NH20H [32-35]. NHZOH-induced Mn extraction occurs only from the lower S-states [155] and results with in conversion of Signal IIv to Signal II f f prOperties similar to those observed upon tris-inhibition. Treatment with various amines [38,39] has also been known to inhibit oxygen evolution activity, acting at the oxidizing side of PSII. Inhibition by ammonia and methylamine appears to be rather direct and does not result in the release of bound Mn that occurs with high concentrations of Tris buffer. Velthuys [40] and Frasch -l49- and Cheniae [31] proposed that water binds to manganese in the OEC by a Lewis acid-—Lewis base mechanism and that amines, being better Lewis bases, are able to compete with H20 for binding sites (see also [9]). According to the model proposed by Velthuys [40], in the initial state 51' no binding of NH occurs; in state 3 52 the binding of ammonia is rapid (half-time about 0.5 s) and rapidly reversible; in state S3 the binding is slower (half-time about 10 s) and slowly reversible. NH3 bound to S4 prevents the oxidation of water. Yocum and Babcock [157] have shown that the block at the $2 or S3 states of the oxygen evolving system by NH3 is sufficient to induce Signal IIf, and more important, has enabled them to demonstrate that retention of Mn(II) in the OEC produces a condition whereby Signal IIf cannot be microwave power saturated. Recently, Sandusky et al. [41], using PSII prepara- tions, have shown that incubation with high concentrations of Tris buffer or NHZOH releases two polypeptides which have been reported to be important for OZ-evolution, the 17 and the 23 KD [42,161,162]. In the same work [41], the authors have presented a series of other treatments (high salt, high pH, etc.) which inhibit OZ-evolution. A selective extraction of the above two polypeptides, but without releasing functional manganese, was possible by using the high salt (pH:6.0) treatment. ~150- By monitoring the ESR properties of 2?, we have explored the role of manganese and of polypeptides, with molecular weights of 17 and 23 KD, in the OZ-evolving process. According to the results presented in this work, the 17 and 23 polypeptides seem to have a structural rather than a catalytic role in 02 evolution activity. Removal of these polypeptides results in a condition whereby exogenous donors, such as benzidine, have ready access to components involved in the water splitting process. B. Materials and Methods Market spinach was used for preparing Chloroplasts with high 02 rates [163]. Leaves were kept dark at 4°C prior to use, then washed in distilled water and deveined under low light conditions. They were broken in a Waring blender for 12 s in a standard solution containing 0.4 M NaCl, 20 mM HEPES, 2 mM MgCl 1 mM EDTA and 2 mg/ml BSA, 2, pH adjusted to 7.5. The homogenate was then strained through 8 layers of cheesecloth and centrifuged for 2 min (5000 r.p.m., SS-34 Sorvall rotor) at about 4°C. The pellets were resuspended in a solution containing 0.15 M NaCl, 4 meMgCl and 20 mM HEPES, pH adjusted to 2 7.5, and then recentrifuged for 2 min (7500 r.p.m.) at about 4°C. The washed samples were resuspended in a minimal volume of SHN (0.4 M sucrose, 10 mM NaCl and 50 mM HEPES buffer) at pH 7.5. Chlorophyll concentrations -151- ranged between 3 and 5 mg chlorophyll per ml as determined by the Sun and Sauer method [94]. 20 ug/ml spinach ferredoxin and 5)<10-4 M NADP, obtained from Sigma, were added to the final chloroplast suspension as an electron acceptor system. Hydroxylamine inactivation was carried out as described in [155]. Photosystem II oxygen evolving particles were prepared as described in Chapter V. A mixture of ferri— cyanide and ferrocyanide was used as an electron acceptor system in PSII particles. "High salt, low pH" inactivation was carried out by incubation of PSII particles in a solution containing 50 mM MES and 2 M NaCl, pH adjusted to 6.0, for 1 hr in the dark at 4°C. For "very low pH" inactivation PSII particles were incubated, for 1 hr in the dark at 4°C, in a solution containing 50 mM Succinate and 10 mM NaCl, pH adjusted to 4. "High salt, high pH" inactivation was carried out by incubation, under the same conditions, in a solution containing 50 mM CAPS buffer and 10 mM NaCl at pH ~10. All the above treatments were followed by a wash with SHN and final resuspension in a minimal volume of SHN (pH:7.5). C. Results It was shown previously that upon extraction of manganese from the oxidizing side of Photosystem II, with the use of Tris buffer, full Signal II magnitude f is observed [116] (the ESR signal of Zip, 21./P680331 [92]). -152- The lifetime of the radical, Zt, is extended into the hundreds of ms time range in the absence of added reductants and is decreased upon addition of exogenous donors (e.g. ascorbate, benzidine) [36] or lipophilic anions (e.g. CCCP) [101] (see also Chapters II and IV). Another treatment which releases functional manganese from OEC is incubation with higher concentrations of NHZOH [32-35]. We studied the behavior of Signal II after NHZOH inactivation and found that full Signal IIf magnitude is observed only when the NHZOH is removed carefully after the inhibitory treatment. It has been shown in Chapter V (see also Ref. [71]) that NH OH is 2 able to inhibit between Z and P680° In the present work we have avoided this complication by a washing procedure which removes NHZOH once its inhibition at the OEC has gone to completion. When these precautions are taken the behavior of Z? parallels that observed in tris- inhibited chloroplasts. Typical data are shown in Fig. 30. The major features can be summarized as follows: (a) 27 formation in the ms range is stoichio- metric with P680; (b) the decay is monophasic and (c) exogenous donors are effective in accelerating its decay. In Fig. 31, we present data on the second order rate constants for benzidine and hydroquinone as donors. The 6 1 -1 rate constants observed, k(BZ)==l.l><10 M- s and 5 l -1 k(HQ)==3.2>100 mW) microwave power saturation properties and -166- follows a biphasic decay time course following illumina- tion. Z? under these conditions is not accessible to exogenous donors. c) Class C: i) High salt incubation at pH 6 and ii) incubation at low pH (~4). The above treatments remove the 17 and 23 KD polypeptides (pH:4.0 releases also the 33 KD, Ghanotakis, D.F. and Yocum, C.F., unpublished observations) but do not alter manganese content. Again, we observe only a fraction of Signal IIf and the species exhibits high (>100 mW) microwave power saturation properties. Z? is apparently accessible to donors like benzidine although the process does not follow the simple first order dependence we observed above for Class A treatments. Rather it appears as if the donor reduces a membrane component, possibly Mn, which in turn reduces the radical (see Discussion). D. Discussion At high NH concentration electron donation by water 3 to Photosystem II is inhibited [38,39]. Velthuys [40,158] has proposed a model in which NH binds to the S2 and 3 53 states but not to S0 and 51' According to this model, in a series of flashes Z? is rereduced by the various S—states as follows (from Ref. 158): -167- k s +zT—1—+S +2 (6.1) 0 1 k + 2 Sl-+Z ———+Sz-+Z 3s'1NH3 52(NH3) (6.2) S (NH )+z‘-‘——+k3 5 (NH )+z 2 3 3 3 0.lorls-l NH3 S3(NH3)2 (6.3) S (NH )-+ZIJEL+S (NH )-+2 (6 4) 3 3 4 3 ° S (NH )+z‘-[—}-<—5—+S (NH) +2 (6 5) 3 32 4 32 ' As shown in the above model S0 and 81 do not bind NH3, 2 and 83 have the ability to bind one and two molecules of NH3 respectively. Studying the oxygen whereas 8 yield in the presence of NH4C1, Delrieu [160] proposed that the turnover times SO+Sl, Sl+S2 and SZ+S3 are accelerated in the presence of NH3, but the 83+S4+S0 reaction is slowed down significantly compared to the respective transition in OZ-evolving systems. From the data presented in references 67 and 160 we conclude that the reduction of Z? in reactions (6.l)-(6.3) is in the usec region, and thus it is difficult to follow by our apparatus, whereas reduction by Eqs. (6.4) and (6.5) is extended into the ms time range and consequently detectable by EPR kinetic techniques. The above model would explain our kinetic results in the presence of NH3 -l68- as follows: i) the fact that only 0.3 spins of Z? are observed in the presence of NH could be explained by 3 assuming that in a steady state experiment (series of 100-200 flashes) only 30% of Z? is rereduced by reactions (6.4) and (6.5). ii) The biphasic decay of Signal IIf would reflect the difference between k4 and k5. As shown in Fig. 32a the overall decay of Z? is a few hundreds of msec (~500 ms), in the absence of Ca2+, which is in good agreement with the value reported in Ref. 160 for the transition S3+SO (~300 ms). The fact that Z? is not accessible to exogenous reductants, like benzidine, could be due to the selectivity of the system in allowing only compounds of certain size and shape to enter the oxidizing side of PSII (see below). In contrast to Tris or NHZOH treatment which removes both, the 17 and the 23 KD polypeptides and functional manganese, high salt (pH:6.0) and low pH (~4.0) treatments remove the above two polypeptides without effecting manganese. Like NH3-treatment, only partial signal is observed and the decay is biphasic. After the above treatments 27 is apparently accessible to donors like benzidine although the process does not follow the simple first order dependence observed for Tris or NHZOH treatment. The fact that the benzidine effect disappears upon addition of NH3 implies that some form 0 I + of manganese could serve as an intermediate between z- and the exogenous donor. Since manganese is in close -169- interaction with 2?, as shown by the power saturation properties of Signal IIf, we postulate that a set of modified S-states (let us call them M-states), which could probably reflect various oxidation states of a manganese complex, serves as a donor system to 2?. The partial Signal IIf observed, could be the result of a mechanism similar to that discussed above for NH3 treatment; low M-states rereduce Z? in very fast reactions, whereas rereduction of Z? by the higher M- states is much slower. M-states, after the removal of the 17 and the 23 KD polypeptides, are accessible to benzidine which converts higher M—states to the lower ones, which in turn rereduce 2?. This model predicts that in the presence of benzidine the kinetic behavior of 27, in a series of successive flashes will depend on the dark time between flashes (td). Long td's will allow benzidine to convert the higher M-states to the lower ones which are going to accelerate the decay of 2?; short td's will not allow such fast conver- sion and thus the benzidine effect will be less dramatic. Indeed, as shown in Fig. 34, variation of the time between flashes (td) changes the decay time of the Z? radical. Longer times between flashes allow the conversion of higher M-states to the lower ones, which results in a faster reduction of Zt. Another model which would explain our results in the presence of NH3 or after extraction of the 17 and 23 KD -l70- polypeptides (with high salt (pH:6.0) or low pH (~4.0) treatment) is the two parallel donor system (see Chapter I and Ref. 8) shown below: S 0 (3) (ii) Z1 P680 S2 (3. \\\£iiL\ Z y2 S 3 + According to this model after photoexcitation P680 is rereduced by two competing donors, 21’ of unknown identity (possibly manganese) and 22, the precursor to Signal IIVf in Oz-evolving chloroplasts and to Signal IIf in inhibited preparations. When both donors are functional only 0.3 spins of z: (or Z?) are observed, whereas upon manganese extraction (with Tris or NHZOH), 22 becomes the only donor to P680 and thus full Signal IIf is observed. If NH3-treatment and polypeptide extraction disturb only reactions (iii) and (iv), the results of Figs. 32-34 would be explained by this model. This model would also explain the fact that Babcock et a1. [67] observed no Signal IIvf for the transitions SO+S1 and Sl+S2 (first two flashes), which -171- at that point was explained by proposing a very fast rereduction of Z+ by states S0 and 81. If we compare the treatments of the three classes presented above (A, B and C) in terms of the accessibility of the oxidizing side of PSII to exogenous donors, we conclude that the 17 and 23 KD polypeptides should have a structural role. When both polypeptides are there neither manganese (S-states) nor 2* is accessible to exogenous donors (Class B). When the polypeptides are extracted but without releasing manganese, exogenous donors react with manganese which in turn reacts with Z? (Class C). When both the polypeptides and manganese are extracted the system becomes wide open and exogenous donors react directly with Z? (Class A). Recently, Radmer and Ollinger [159] proposed that the water binding site resides in a cleft which allows selectively the entrance of molecules with certain size and shape. 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