Concentration-dependent effects of salicylaldoxime on chloroplast reactions

Salicylaldoxime has been found to have a variety of concentration-dependent effects on chloroplast activities. At low concentrations (< 10 mM), salicylaldoxime reversibly inhibits all reactions which involve Photosystem II. Since the DCMU-insensitive silicomolybdate Hill reaction is also inhibited, one site of inhibition is definitely located before the DCMU-sensitive site, possibly before the photoact. The inhibition kinetics and the response of chloroplast fluorescence may indicate another site in the DCMU-sensitive region. At almost exactly the same concentrations (< 10 mM), salicylaldoxime uncouples phosphorylation reversibly, whether it is supported by Photosystem II or by Photosystem I. At higher concentrations (approx. 20 mM) salicylaldoxime inhibits Photosystem II irreversibly, uncouples irreversibly, and begins to cause changes in chloroplast light scattering which could be manifestations of membrane damage. At very high concentrations (approx. 45 mM) salicylaldoxime irreversibly inhibits Photosystem I activity in the region of plastocyanin. This is indicated by the ability of salicylaldoxime to inhibit the photooxidation of cytochrome f but not the photooxidation of P-700.     

The effects of high concentrations of salts on photosynthetic electron transport in spinach (Spinacia oleracea) chloroplasts.

1. Photosynthetic electron transport from water to lipophilic Photosystem II acceptors was stimulated 3--5-fold by high concentrations (greater than or equal to 1 M) of salts containing anions such as citrate, succinate and phosphate that are high in the Hofmeister series. 2. In trypsin-treated chloroplasts, K3Fe(CN)6 reduction insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea was strongly stimulated by high concentrations of potassium citrate, but there was much less stimulation of 2,6-dichloroindophenol reduction in Tris-treated chloroplasts supplied with 1,5-diphenylcarbazide as artificial donor. The results suggest that the main site of action of citrate was the O2-evolving complex of Photosystem II. 3. Photosystem I partial reactions were also stimulated by intermediate concentrations of citrate (up to 2-fold stimulation by 0.6--0.8 M-citrate), but were inhibited at the highest concentrations. The observed stimulation may have been caused by stabilizaton of plastocyanin that was complexed with the Photosystem I reaction centre, 4. At 1 M, potassium citrate protected O2 evolution against denaturation by heat or by the chaotropic agent NaNO3. 5. It is suggested that anions high in the Hofmeister series stimulated and stabilized electron transport by enhancing water structure around the protein complexes in the thylakoid membrane.     

The effect of ethylenediamine chemical modification of plastocyanin on the rate of cytochrome f oxidation and P-700+ reduction

Chemical modification of plastocyanin was carried out using ethylenediamine plus a water-soluble carbodiimide, which has the effect of replacing a negatively charged carboxylate group with a positively charged amino group at pH 6-8. The conditions were adjusted to produce a series of singly and doubly modified forms of plastocyanin. Differences in charge configuration allowed separation of these forms on a Pharmacia fast protein liquid chromatograph using a Mono Q anion exchange column. These forms were used to study the interaction of plastocyanin with its reaction partner cytochrome f. The rate of cytochrome f oxidation was progressively inhibited upon incorporation of increasing numbers of ethylenediamine moieties indicating a positively charged binding site on cytochrome f. However, differential inhibition was obtained for the various singly modified forms allowing mapping of the binding site on plastocyanin. The greatest inhibition was found for forms modified at negatively charged residues Nos. 42-45 and Nos. 59-61 which comprise a negative patch surrounding Tyr-83. In contrast, the form modified at residue No. 68, on the opposite side of the globular plastocyanin molecule, showed the least inhibition. It can be concluded that the binding site for cytochrome f is located in the vicinity of residues Nos. 42-45 and Nos. 59-61. Modification of plastocyanin at residues Nos. 42-45 showed no effect on the rate of P-700+ reduction, suggesting that these residues are not involved in the binding of Photosystem I. However, an increase in the rate of P-700+ reduction was observed for plastocyanins modified at residue No. 68 or Nos. 59-61, which is consistent with the idea that the reaction domain of Photosystem I is negatively charged and Photosystem I binds at the top of the molecule and accepts electrons via His-87 in plastocyanin. These results raise the possibility that plastocyanin can bind both cytochrome f and Photosystem I simultaneously. The effect of ethylenediamine modification on the formal potential of plastocyanin was also examined. The formal potential of control plastocyanin was found to be +372 +/- 5 mV vs. normal hydrogen electrode at pH 7. All modified forms showed a positive shift in formal potential. Singly modified forms showed increases in formal potentials between +8 and +18 mV with the largest increases being observed for plastocyanins modified at residues Nos. 42-45 or Nos. 59-61.     

The effect of ethylenediamine chemical modification of plastocyanin on the rate of cytochrome f oxidation and P-700 reduction

Chemical modification of plastocyanin was carried out using ethylenediamine plus a water-soluble carbodiimide, which has the effect of replacing a negatively charged carboxylate group with a positively charged amino group at pH 6–8. The conditions were adjusted to produce a series of singly and doubly modified forms of plastocyanin. Differences in charge configuration allowed separation of these forms on a Pharmacia fast protein liquid chromatograph using a Mono Q anion exchange column. These forms were used to study the interaction of plastocyanin with its reaction partner cytochrome f. The rate of cytochrome f oxidation was progressively inhibited upon incorporation of increasing numbers of ethylenediamine moieties indicating a positively charged binding site on cytochrome f. However, differential inhibition was obtained for the various singly modified forms allowing mapping of the binding site on plastocyanin. The greatest inhibition was found for forms modified at negatively charged residues Nos. 42–45 and Nos. 59–61 which comprise a negative patch surrounding Tyr-83. In contrast, the form modified at residue No. 68, on the opposite side of the globular plastocyanin molecule, showed the least inhibition. It can be concluded that the binding site for cytochrome f is located in the vicinity of residues Nos. 42–45 and Nos. 59–61. Modification of plastocyanin at residues Nos. 42–45 showed no effect on the rate of P-700⁺ reduction, suggesting that these residues are not involved in the binding of Photosystem I. However, an increase in the rate of P-700⁺ reduction was observed for plastocyanins modified at residue No. 68 or Nos. 59–61, which is consistent with the idea that the reaction domain of Photosystem I is negatively charged and Photosystem I binds at the top of the molecule and accepts electrons via His-87 in plastocyanin. These results raise the possibility that plastocyanin can bind both cytochrome f and Photosystem I simultaneously. The effect of ethylenediamine modification on the formal potential of plastocyanin was also examined. The formal potential of control plastocyanin was found to be +372 ± 5 mV vs. normal hydrogen electrode at pH 7. All modified forms showed a positive shift in formal potential. Singly modified forms showed increases in formal potentials between +8 and +18 mV with the largest increases being observed for plastocyanins modified at residues Nos. 42–45 or Nos. 59–61.     

Contrasting effects of plastocyanin on the photoreduction and photooxidation of cytochrome f in chloroplasts

Removal of plastocyanin from Photosystem I subchloroplast particles had no effect on the Photosystem I photooxidation of cytochrome f. Chloroplasts depleted of plastocyanin by sonication lost the ability to reduce cytochrome f in Photosystem II light. Addition of plastocyanin restored the photoreduction of cytochrome f. These results are consistent with a plastocyanin site on the reducing side of cytochrome f.     

Proton translocation in chloroplasts and its relationship to electron transport between the photosystems.

Using dark adapted isolated spinach chloroplasts and sequences of brief saturating flashes the correlation of the uptake and release of protons with electron transport from Photosystem II to Photosystem I were studied. The following observations and conclusions are reported: (1) Flash-induced proton uptake shows a weak, damped binary oscillation, with maxima occurring after the 2nd, 4th, etc. flashes. The damping factor is comparable to that observed in the O2 flash yield oscillation and therefore explained by misses in Photosystem II. (2) On the average and after a steady state is reached, each flash (i.e. each reduction of Q) induces the uptake of 2H+ from outside the chloroplasts. (3) Flash induced proton release inside the chloroplast membrane shows a strong damped binary oscillation with maximum release occurring also after the 2nd, 4th, etc. flashes. (4) This phenomenon is correlated with the earlier reported binary oscillations of electron transport [2] and shows that both electrons and protons are transported in pairs between the photosystems. (5) In two sequential flashes 4H+ from the outside of the thylakoid and 2e- from water are accumulated at a binding site B. Subsequently, the two electrons are transferred to non-protonated acceptors in Photosystem I (probably plastocyanin and cytochrome f) and the 4H+ are released inside the thylakoid. (6) It is concluded that a primary proton transporting site and/or energy conserving step located between the photosystems is being observed.     

Reactions of plastocyanin and cytochrome 553 with Photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates.

It is suggested that besides the reaction sequence cytochrome 553 → plastocyanin → Photosystem I reaction center, a second pathway cytochrome 553 → Photosystem I reaction center may operate additionally.     

Lipophilicity and catalysis of photophosphorylation. II. Quinoid compounds as artificial carriers in cyclic photophosphorylation and photoreductions by Photosystem I

The topography of the chloroplast membrane has been studied using the following pairs of quinoid compounds with similar structure and chemical properties, but with different lipid solubility: phenazine/sulfophenazine, naphthoquinone/naphthoquinone sulfonate, indophenol/sulfoindophenol and lumiflavin/FMN.

All these compounds in the oxidized form are able to accept electrons from the photosynthetic electron transport chain in Hill reactions. However, only the lipophilic compounds in the reduced form can donate electrons to Photosystem I, when electron flow from Photosystem II is blocked by inhibitors. This is in agreement with the notation that the oxidizing site of Photosystem I (P₇₀₀⁺) and the electron donors for Photosystem I (cytochrome f and plastocyanin) are located inside the lipid barrier of the inner chloroplast membrane. The reducing sites in the Hill reactions must be located on the outer surface, accessible from the suspending medium.

It has been known for a long time that N,N′-tetramethyl-p-phenylenediamine can donate electrons to Photosystem I, but contrary to diaminodurene (2,3,5,6-tetramethyl phenylenediamine) it does not induce ATP formation. Both compounds are lipophilic and have similar redox potentials, but only the latter carries hydrogens which are involved in the redox reaction. For energy conservation, coupled to electon flow in Photosystem I, it therefore seems necessary that the lipophilic redox compound in the reduced form can carry hydrogens through the chloroplast membrane.     

Mechanism of KCN inhibition of photosystem I.

Experiments with chloroplasts and purified spinach plastocyanin suggest a mechanism for KCN inhibition of Photosystem I. KCN inhibition can be bypassed by a detergent or reversed by replacement of the inactive plastocyanin. KCN bleaches and inactivates purified plastocyanin. KCN releases copper from chloroplast membranes and from purified plastocyanin. Cyanide does not bind to the apoprotein produced when plastocyanin is treated with KCN, and KCN-produced apoplastocyanin has a N-ethylmaleimide-reactive sulfhydryl group not found in holoplastocyanin. Apoplastocyanin is not active in restoring Photosystem I activity to plastocyanin-depleted membranes. Holoplastocyanin restores Photosystem I activities to plastocyanin-depleted membranes prepared from either control or KCN-treated chloroplasts to about the same extent. KCN-treated chloroplast membranes are found to have higher amounts of apoplastocyanin than do control chloroplast membranes. These results offer evidence that KCN removes the copper from plastocyanin in the chloroplast membrane, leaving the inactive apoplastocyanin which is unable to transfer electrons to Photosystem I.     

Transient kinetics of the electron transfer between P-700, plastocyanin and cytochrome f in chloroplasts suspended in fluid media at sub-zero temperatures

The kinetics of redox changes of P-700, plastocyanin and cytochrome f in chloroplasts suspended in a fluid medium at sub-zero temperatures have been studied following excitation of the chloroplasts with either a single-turnover flash, a series of flashes or continuous light. The results show that: (1) The kinetics of reduction of P-700⁺ and those of oxidation of plastocyanin are consistent with a bimolecular reaction between these two components as previously suggested (Olsen, L.F., Cox, R.P. and Barber, J. (1980) FEBS Lett. 122, 13–16). (2) Cytochrome f shows heterogeneity with respect to its kinetics of oxidation by Photosystem I. (3) In contrast to the situation when plastoquinol is the electron donor, reduction of cytochrome f by electrons derived from diaminodurene occurs with sigmoidal kinetics that shows a good fit to an apparent equilibrium constant of 12 between the cytochrome and P-700. (4) The rate of electron transfer from plastoquinol to Photosystem I depends on the redox state of the plastoquinone pool. (5) In relation to current ideas about the lateral heterogeneity of Photosystem I and Photosystem II in the thylakoid membrane, the results are consistent with the function of plastocyanin as a mobile carrier of electrons in the intrathylakoid space.     

Genetic variation in soybean photosynthetic electron transport capacity is related to plastocyanin concentration in the chloroplast

Fifteen ancestral genotypes of United States soybean cultivars were screened for differences in photosynthetic electron transport capacity using isolated thylakoid membranes. Plants were grown in controlled environment chambers under high or low irradiance conditions. Thylakoid membranes were isolated from mature leaves. Photosynthetic electron transport was assayed as uncoupled Hill activity using 2,6-dichlorophenolindophenol (DCIP). Soybean electron transport activity was dependent on genotype and growth irradiance and ranged from 6 to 91 mmol DCIP reduced [mol chlorophyll]^–1 s^–1. Soybean plastocyanin pool size ranged from 0.1 to 1.3 mol plastocyanin [mol Photosystem I]^–1. In contrast, barley and spinach electron transport activities were 140 and 170 mmol DCIP reduced [mol chlorophyll]^–1 s^–1, respectively, with plastocyanin pool sizes of 3 to 4 mol plastocyanin [mol Photosystem I]^–1. No significant differences in the concentrations of Photosystem II, plastoquinone, cytochrome b₆f complexes, or Photosystem I were observed. Thus, genetic differences in electron transport activity were correlated with plastocyanin pool size. The results suggested that plastocyanin pool size can vary significantly and may limit photosynthetic electron transport capacity in certain species such as soybean. Soybean plastocyanin consisted of two isoforms with apparent molecular masses of 14 and 11 kDa, whereas barley and spinach plastocyanins each consisted of single polypeptides of 8 and 12 kDa, respectively.Abbreviations DAP days after planting - DCIP 2,6-dichlorophenolindophenol - LiDS lithium dodecyl sulfate - PPFD photosynthetic photon flux density (mol photons m^–2 s^–1) - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Polylysine-enhanced effectiveness of plastocyanin in photosystem I

1. Chloroplast fragments from either Chlamydomonas reinhardi or spinach, which lack plastocyanin, or from Euglena gracilis depleted of cytochrome c552, require a large excess of exogenously added plastocyanin or cytochrome c552 to restore Photosystem I activity.2. In the presence of a small amount of polylysine, Photosystem I activity of chloroplast fragments is stimulated greatly by plastocyanin or cytochrome c552, and the reaction is saturated at a lower concentration of these proteins. Higher concentrations of polylysine inhibit Photosystem I activity; the inhibition is not reversed by plastocyanin or cytochrome c552.3. Salt protects chloroplast fragments from stimulation by polylysine plus plastocyanin or cytochrome c552, and also reverses this stimulation.4. The data suggest that polylysine, at low concentration, enhances binding of plastocyanin or cytochrome c552 to chloroplast membranes, thereby increasing the effective concentration at their site of function. The total inhibition of Photosystem I activity, independent of the presence of plastocyanin or cytochrome c552, at higher polylysine concentrations is similar probably to that observed previously in chloroplasts which retain their plastocyanin.     

Pathways of silicomolybdate photoreduction and associated photophosphorylation in tobacco chloroplasts.

Three sites of silicomolybdate reduction in the electron transport chain of isolated tobacco chloroplasts are described. The relative participation of these sites is greatly influenced by the particular reaction conditions. One site (the only site when the reaction medium contains high concentrations of bovine serum albumin (greater than 5 mg/ml) is associated with Photosystem I, since it supports phosphorylation with a P/e2 value close to 1 and the reaction is totally sensitive to both plastocyanin inhibitors and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Two other sites of silicomolybdate reduction are associated with Photosystem II. One site is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive and supports phosphorylation when the reaction mixture contains dimethyl sulfoxide and glycerol (protective agents). The P/e2 value routinely observed is about 0.2. Bovine serum albumin (1-2 mg/ml) can also act as a protective agent, but the efficiency of Photosystem II phosphorylation observed is lower. Silicomolybdate reduction supports virtually no phosphorylation, regardless of the reduction pathway, when the reaction mixture contains no protective agents. This is due to irreversible uncoupling by silicomolybdate itself. The silicomolybdate uncoupling is potentiated by high salt concentrations even if the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.     

Pathways of silicomolybdate photoreduction and the associated photophosphorylation in tobacco chloroplasts

Three sites of silicomolybdate reduction in the electron transport chain of isolated tobacco chloroplasts are described. The relative participation of these sites is greatly influenced by the particular reaction conditions. One site (the only site when the reaction medium contains high concentrations of bovine serum albumin (> 5 mg/ml)) is associated with Photosystem I, since it supports phosphorylation with a P/e₂ value close to 1 and the reaction is totally sensitive to both plastocyanin inhibitors and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Two other sites of silicomolybdate reduction are associated with Photosystem II. One site is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive and supports phosphorylation when the reaction mixture contains dimethyl sulfoxide and glycerol (protective agents). The P/e₂ value routinely observed is about 0.2. Bovine serum albumin (1–2 mg/ml) can also act as a protective agent, but the efficiency of Photosystem II phosphorylation observed is lower. Silicomolybdate reduction supports virtually no phosphorylation, regardless of the reduction pathway, when the reaction mixture contains no protective agents. This is due to irreversible uncoupling by silicomolybdate itself. The silicomolybdate uncoupling is potentiated by high salt concentrations even in the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.     

The site of KCN inhibition in the photosynthetic electron transport pathway

Treatment of chloroplasts with high concentrations of KCN inhibits reactions which involve Photosystem I (e.g. electron transport from water or diaminodurene to methylviologen), but not those assumed to by-pass Photosystem I (e.g. electron transport from water to quinonediimides). The spectrophotometric experiments described in this paper showed that KCN inhibits the oxidation of cytochrome f by far-red light without blocking its reduction by red light. Both optical and EPR experiments indicated that KCN does not inhibit the photooxidation of P700 but markedly slows down the subsequent dark decay (reduction). Reduction of P700 by Photosystem II is prevented by KCN. It is concluded that KCN blocks electron transfer between cytochrome f and P700, i.e. the reaction step which is believed to be mediated by plastocyanin. In KCN-poisoned chloroplasts the slow dark reduction of P700 following photooxidation is greatly accelerated by reduced 2,6-dichlorophenolindophenol or by reduced N-methylphenazonium methosulfate (PMS), but not by diaminodurene. It appears that the reduced indophenol dye and reduced PMS are capable of donating electrons directly to P700, at least partially by-passing the KCN block.     

Effect of growth irradiance on plastocyanin levels in barley

Plastocyanin levels in barley (Hordeum vulgare cv Boone) were found to be dependent on growth irradiance. An immunochemical assay was developed and used to measure the plastocyanin content of isolated thylakoid membranes. Barley grown under 600 mole photons m^–2s^–1 contained two- to four-fold greater quantities of plastocyanin per unit chlorophyll compared with plants grown under 60 mole photons m^–2s^–1. The plastocyanin/Photosystem I ratio was found to be 2 to 3 under high irradiance compared with 0.5 to 1.5 under low irradiance. The reduced plastocyanin pool size in low light plants contributed to a two-fold reduction in photosynthetic electron transport activity. Plastocyanin levels increased upon transfer of low light plants to high irradiance conditions. In contrast, plastocyanin levels were not affected in plants transferred from high to low irradiance, suggesting that plastocyanin is not involved in the acclimation of photosynthesis to shade.Abbreviations: BSA bovine serum albumin - chl chlorophyll - cyt cytochrome - DCIP 2,6-dichlorophenolindophenol - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I - TBS 20 mM Tris-HCl pH 7.5, 500 mM NaCl - TTBS 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.5% (w/v) polyoxyethylenesorbitan monolaurate (Tween-20)     

Photosynthetic electron transfer through the cytochrome b6f complex can bypass cytochrome f

The cytochrome b(6)f complex is an obligatory electron transfer and proton-translocating enzyme in all oxygenic photosynthesis. Its operation has been described by the "Q-cycle." This model proposes that electrons are transferred from plastoquinol to plastocyanin (the reductant of P700 in Photosystem I) through, obligatorily in series, the iron-sulfur and the cytochrome f redox centers in the cytochrome b(6)f complex. However, here we demonstrate that (a) the iron-sulfur center-dependent reductions of plastocyanin and P700 are much faster than cytochrome f reduction, both in Chlamydomonas reinhardtii cytochrome f mutants and in the wild type, and (b) the steady-state photosynthetic electron transport does not correlate with strongly inhibited cytochrome f reduction kinetics in the mutants. Thus, cytochrome f is not an obligatory intermediate for electrons flowing through the cytochrome b(6)f complex. The oxidation equivalents from Photosystem I are delivered to the high potential chain of the cytochrome b(6)f complex both at the cytochrome f level and, independently, at another site connected to the quinol-oxidizing site, possibly the iron-sulfur center.     

Evidence for multiple sites of ferricyanide reduction in chloroplasts.

Various sites of ferricyanide reduction were studied in spinach chloroplasts. It was found that in the presence of dibromothymoquinone a fraction of ferricyanide reduction was dibromothymoquinone sensitive, implying that ferricyanide can be reduced by photosystem I as well as photosystem II. To separate ferricyanide reduction sites in photosystem II, orthophenanthroline and dichlorophenyl dimethylurea inhibitions were compared at various pHs. It was noted that at low pH ferricyanide reduction was not completely inhibited by orothophenanthroline. At high pH's, however, inhibition of ferricyanide reduction by orthophenanthroline was complete. It was found that varying concentration of orthophenanthroline at a constant pH showed different degrees of inhibition. In the study of ferricyanide reduction by photosystem II various treatments affecting plastocyanin were performed. It was found that Tween-20 or KCN treatments which inactivated plastocyanin did not completely inactivate ferricyanide reduction. These data support the conclusion that ferricyanide accepts electrons both before and after plastoquinone in photosystem II.     

Evidence for multiple sites of ferricyanide reduction in chloroplasts

Various sites of ferricyanide reduction were studied in spinach chloroplasts. It was found that in the presence of dibromothymoquinone a fraction of ferricyanide reduction was dibromothymoquinone sensitive, implying that ferricyanide can be reduced by photosystem I as well as photosystem II. To separate ferricyanide reduction sites in photosystem II, orthophenanthroline and dichlorophenyl dimethylurea inhibitions were compared at various pH's. It was noted that at low pH ferricyanide reduction was not completely inhibited by orthophenanthroline. At high pH's, however, inhibition of ferricyanide reduction by orthophenanthroline was complete. It was found that varying concentration of orthophenanthroline at a constant pH showed different degrees of inhibition. In the study of ferricyanide reduction by photosystem II various treatments affecting plastocyanin were performed. It was found that Tween-20 or KCN treatments which inactivated plastocyanin did not completely inactivate ferricyanide reduction. These data support the conclusion that ferricyanide accepts electrons both before and after plastoquinone in photosystem II.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyurea - MV methyl viologen - DBMIB 2,5-dibromothymoquinone - DMBQ 2,6-dimethyl benzoquinone - OP 1,10-orthophenanthroline - TMPD tetramethyl-p-phenylenediamine - PS 1 photosystem I - PS II photosystem II - SN sucrose-sodium chloride chloroplasts Supported by NSF Grant BMS 74-19689.