Different roles of plastoquinone in the photoreduction of ferredoxin and of membrane-bound iron-sulfur centers of chloroplasts

Photosynthetic electron transport in chloroplasts was inhibited by the plastoquinone antagonist, dibromothymoquinone (DBMIB) in two steps. Lower concentrations of DBMIB inhibited the photoreduction of the bound iron-sulfur centers of photosystem I without inhibiting the photoreduction of ferredoxin. Higher concentrations of DBMIB did inhibit the oxygenic photoreduction (i.e., by water) of ferredoxin and NADP⁺, but their photoreduction was restored, wholly or partly, by each of four chemically diverse uncouplers, similar only in facilitating proton movement across membranes. By contrast, none of the uncouplers alleviated the DBMIB inhibition of the photoreduction of the bound Fe-S centers. These divergent responses to uncouplers are incompatible with the Z scheme but are consistent with the new concept of oxygenic and anoxygenic photosystems in plant photosynthesis (Proc. Natl. Acad. Sci. USA $\text{78}$, 2942–2946, 1981).     

Divergent pathways of photosynthetic electron transfer: The autonomous oxygenic and anoxygenic photosystems

The aim of this article is to assemble and integrate, from a personal perspective of a research participant, seldom examined evidence that is incompatible with some basic tenets of photosynthetic electron transport, the cornerstone of which is the Z scheme. The nonconforming evidence pertaining to the mode of ferredoxin reduction and the role of the copper redox protein, plastocyanin, indicates that contrary to the Z scheme ferredoxin is reduced in two experimentally distinguishable ways: oxygenically by PS II (renamed the oxygenic photosystem), without the participation of PS I, and anoxygenically by PS I (renamed the anoxygenic photosystem). It also indicates that plastocyanin is not only, as the Z scheme asserts, the electron donor to the reaction center chlorophyll of PS I (P700) but also to the reaction center chlorophyll of PS II (P680). Other unconventional findings include evidence that the fully functional oxygenic photosystem, when operating separately from the anoxygenic photosystem, reduces plastoquinone to plastoquinol and subsequently oxidizes plastoquinol by two pathways acting in concert: one being the universally recognized DBMIB-sensitive pathway via the Rieske iron-sulfur center of the cytochrome bf complex and the other, a hitherto unrecognized, DBMIB-insensitive electron transport pathway around P680 that centers on cytochrome b-559. These nonconforming findings form the basis of an alternate hypothesis of photosynthetic electron transport that modifies and complements the Z scheme.Abbreviations PS photosystem - PQ oxidized plastoquinone - PQH₂ reduced plastoquinone (plastoquinol) - Q_A and Q_B specialized membrane-bound forms of PQ - PC plastocyanin - Fd ferredoxin - BISC F_AF_B, membrane-bound iron-sulfur centers of PS I - DBM1B 2,5-dibromo-3-methyl-6-isopropyl-n-benzoquinone (dibromothymoquinone) - DNP-INT dinitrophenol ether of iodonitrothymol - NADP⁺ NADPH, oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate - FCCP carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone - CCCP carbonyl cyanide-3-chlorophenylhydrazone - SF 6847 2,6,-di-(t-butyl)-4-(2,2-dicyanovinyl) phenol - diuron (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - DCIP 2,6-dichloro-phenolindophenol - UHDBT 5-(n-undecyl)-6-hydroxy-4-7-dioxobenzothiazole; cytochrome b-559_HP-cytochrome b-559_LP, high- and low potential states of cytochrome b-559 - oxygenic reductions reductions in which water is the electron donor - BBY PS II preparation made according to Berthold et al. (1981) Dedicated to Professor Achim Trebst on his 65th birthday.Based in part on lecture in Advanced Course on Trends in Photosynthesis Research, Palma de Mallorca, Spain, September 18, 1990.     

Ferredoxin and flavodoxin reduction by photosystem I.

Ferredoxin and flavodoxin are soluble proteins which are reduced by the terminal electron acceptors of photosystem I. The kinetics of ferredoxin (flavodoxin) photoreduction are discussed in detail, together with the last steps of intramolecular photosystem I electron transfer which precede ferredoxin (flavodoxin) reduction. The present knowledge concerning the photosystem I docking site for ferredoxin and flavodoxin is described in the second part of the review.     

Kinetic evidence for the PsaE-dependent transient ternary complex photosystem I/Ferredoxin/Ferredoxin:NADP(+) reductase in a cyanobacterium.

A mutant of Synechocystis PCC 6803, deficient in psaE, assembles photosystem I reaction centers without the PsaE subunit. Under conditions of acceptor-side rate-limited photoreduction assays in vitro (with 15 microM plastocyanin included), using 100 nM ferredoxin:NADP(+) reductase (FNR) and either Synechocystis flavodoxin or spinach ferredoxin, lower rates of NADP(+) photoreduction were measured when PsaE-deficient membranes were used, as compared to the wild type. This effect of the psaE mutation proved to be due to a decrease of the apparent affinity of the photoreduction assay system for the reductase. In the psaE mutant, the relative petH (encoding FNR) expression level was found to be significantly increased, providing a possible explanation for the lack of a phenotype (i.e., a decrease in growth rate) that was expected from the lower rate of linear electron transport in the mutant. A kinetic model was constructed in order to simulate the electron transfer from reduced plastocyanin to NADP(+), and test for possible causes for the observed change in affinity for FNR. The numerical simulations predict that the altered reduction kinetics of ferredoxin, determined for the psaE mutant [Barth, P., et al., (1998) Biochemistry 37, 16233-16241], do not significantly influence the rate of linear electron transport to NADP(+). Rather, a change in the dissociation constant of ferredoxin for FNR does affect the saturation profile for FNR. We therefore propose that the PsaE-dependent transient ternary complex PSI/ferredoxin/FNR is formed during linear electron transport. Using the yeast two-hybrid system, however, no direct interaction could be demonstrated in vivo between FNR and PsaE fusion proteins.     

Effects of magnesium and chloride ions on light-induced electron transport in membrane fragments from a blue-green alga

The effects of magnesium and chloride ions on photosynthetic electron transport were investigated in membrane fragments of a blue-green alga, Nostoc muscorum (Strain 7119), noted for their stability and high rates of electron transport from water or reduced dichlorophenolindophenol to NADP⁺. Magnesium ions were required not only for light-induced electron transport from water to NADP⁺ but also for protection in the dark of the integrity of the water-photooxidizing system (Photosystem II). Membrane fragments suspended in the dark in a medium lacking Mg²⁺ lost the capacity to photoreduce NADP⁺ with water on subsequent illumination. Chloride ions could substitute, but less effectively, for each of these two effects of magnesium ions. By contrast, the photoreduction of NADP⁺ by DCIPH₂ was independent of Mg²⁺ (or Cl^?) for the protection of the electron transport system in the dark or during the light reaction proper. Furthermore, high concentrations of MgCl₂ produced a strong inhibition of NADP⁺ photoreduction with DCIPH₂ without significantly affecting the rate of NADP⁺ photoreduction with water. The implications of these findings for the differential involvement of Photosystem I and Photosystem II in the photoreduction of NADP⁺ with different electron donors are discussed.     

Evaluation of photosynthetic activities in thylakoid membranes by means of Fourier transform infrared spectroscopy

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn₄CaO₅ cluster and Q_B reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to Q_A reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of Q_A^? and P700⁺, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.     

Interaction between photosystem I and ferredoxin. Identification by chemical cross-linking of the polypeptide which binds ferredoxin

Ferredoxin has been effectively cross-linked to photosystem I complex by treatment of purified particles or thylakoids with N-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker which stabilizes protein-protein electrostatic interactions. Analysis of photosystem I polypeptide composition after such a treatment showed a specific decrease of the 20-kDa subunit and the appearance of a new component of about 42 kDa which was recognized by the anti-ferredoxin antibody. Cross-linking of ferredoxin to thylakoids allowed the membrane preparation to photoreduce cytochrome c without requiring exogenous ferredoxin, whereas photosystem I particles purified from treated thylakoids were inactivated in the NADP+ photoreduction activity. From these results, it can be inferred that the polypeptide of 20 kDa is the photosystem I subunit which interacts with ferredoxin during the photosynthetic electron transport.     

Photochemical activity and components of membrane preparations from blue-green algae. I. Coexistence of two photosystems in relation to chlorophyll a and removal of phycocyanin

Nostoc muscorum (Strain 7119) cells were disrupted and the accessory pigment phycocyanin was removed from membrane fragments by digitonin treatment. The phycocyanin-depleted membrane fragments retained both Photosystem I and Photosystem II activity, as evidenced by high rates of NADP⁺ photoreduction either by water or by reduced 2,6-dichlorophenolindophenol, indicating that phycocyanin is not an essential component for electron transport activity.No separation of the two photosystems was effected by the digitonin treatment. Even drastic digitonin treatments failed to diminish significantly the remarkably stable electron transport from water to NADP⁺.Action spectra and relative quantum efficiency measurements demonstrated the existence of both Photosystem I and Photosystem II in membrane fragments which contained chlorophyll a as the only significant light-absorbing pigment.     

Crystallization and electron paramagnetic resonance characterization of the complex of photosystem I with its natural electron acceptor ferredoxin

The formation of a transient complex between photosystem I and ferredoxin is involved in the process of ferredoxin photoreduction in oxygenic photosynthetic organisms. Reduced ferredoxin is an essential redox intermediate involved in many assimilatory processes and is necessary for the reduction of NADP(+) to NADPH. Single crystals from a complex of photosystem I with ferredoxin were grown using PEG 400 and CaCl(2) as precipitation agents. The crystals diffract x-rays to a resolution of 7-8 A. The space group was determined to be orthorhombic with the unit cell dimensions a = 194 A, b = 208 A, and c = 354 A. The crystals contain photosystem I and ferredoxin in a 1:1 ratio. Electron paramagnetic resonance (EPR) measurements on these crystals are reported, where EPR signals of the three [4Fe-4S] clusters F(A), F(B), F(X), and the [2Fe-2S] cluster of ferredoxin were detected. From the EPR spectra observed at three particular orientations of the crystal in the magnetic field, the full orientation pattern of the F g-tensor was simulated. This simulation is consistent with the presence of 12 magnetically inequivalent F clusters per unit cell with the C(3) axis of the PSI trimers oriented at (23 degrees, 72 degrees, 77 degrees ) to the unit cell axes.     

Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii: (II) Involvement of the PGR5–PGRL1 pathway under anaerobic conditions

In oxygenic photosynthesis, cyclic electron flow around photosystem I denotes the recycling of electrons from stromal electron carriers (reduced nicotinamide adenine dinucleotide phosphate, NADPH, ferredoxin) towards the plastoquinone pool. Whether or not cyclic electron flow operates similarly in Chlamydomonas and plants has been a matter of debate. Here we would like to emphasize that despite the regulatory or metabolic differences that may exist between green algae and plants, the general mechanism of cyclic electron flow seems conserved across species. The most accurate way to describe cyclic electron flow remains to be a redox equilibration model, while the supramolecular reorganization of the thylakoid membrane (state transitions) has little impact on the maximal rate of cyclic electron flow. The maximum capacity of the cyclic pathways is shown to be around 60 electrons transferred per photosystem per second, which is in Chlamydomonas cells treated with 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and placed under anoxic conditions. Part I of this work (aerobic conditions) was published in a previous issue of BBA-Bioenergetics (vol. 1797, pp. 44–51) (Alric et al., 2010).     

Inhibition of electron flow around photosystem I in chloroplasts of Cd-treated maize plants is due to Cd-induced iron deficiency

Photosystem I activity of chloroplasts isolated from 21 days old maize seedlings ( Zea mays L. cv. Hidosil) cultivated in a nutrient solution containing different concentrations of Cd (10,20,30μM) was investigated. Cd markedly decreased ferredoxin(Fd)-dependent NADP⁺ photoreduction, while it had no effect on electron transport from 2. 6-dichlorophenolindophenol to methyl viologen, indicating that the metal interferred with electron transport on the reducing side of photosystem I. The decrease in electron transport correlated with a low Fd content, which in turn was correlated with a low Fe concentration, suggesting Cd-induced Fe deficiency. In in vitro experiments direct Cd inhibition of Fd-dependent NADP⁺ photoreduction required much higher Cd concentrations than those observed in Cd-treated plants.     

EPR spectra of Photosystem I and other iron protein components in intact cells of cyanobacteria

Electron paramagnetic resonance (EPR) spectra were recorded of whole filaments of the cyanobacteria Nostoc muscorum and Anabaena cylindrica. Signals due to manganese were removed by freezing and thawing the cells in EDTA. EPR spectra were assigned on the basis of their g values, linewidths, temperature dependence and response to dithionite and light treatments. The principal components identified were: (i) rhombic Fe³⁺ (signal at g = 4.3), probably a soluble storage form of iron; (ii) iron-sulfur centers A and B of Photosystem I; (iii) the photochemical electron acceptor ‘X’ of Photosystem I; this component was also observed for the first time in isolated heterocysts; (iv) soluble ferredoxin which was present at a concentration of 1 molecule per 140 ± 20 chlorophyll molecules; (v) a membrane-bound iron-sulfur protein (g = 1.92). A signal g = 6 in the oxidized state was probably due to an unidentified heme compound. During deprivation of iron the rhombic Fe³⁺, centers A, B and X of Photosystem I, and soluble ferredoxin were all observed to decrease.     

The relationship of the cyclic and non-cyclic electron transport pathways in chloroplasts

Light-induced redox changes of plastocyanin, the Rieske iron-sulfur center, and P-700 have been studied in situ in spinach chloroplasts. Plastocyanin and the Rieske center behaved in an analogous manner in that their steady states were fully oxidized in the light in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when an electron acceptor is present. After illumination under conditions of non-cyclic electron transfer from water to an electron acceptor, followed by a short dark period, the steady state of both shifted to a more reduced level. A 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive photoreduction of the Rieske center was observed in ferricyanide-washed chloroplast fragments. With reduced ferredoxin as electron donor, it was possible to demonstrate a reduction in the dark of these electron carriers and of P-700; this reduction was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea but was inhibited by antimycin A. These findings are discussed in relation to a function for these electron carriers in the cyclic electron transport pathway in chloroplasts and to their function in the non-cyclic electron transport pathway.     

Diazonium modification of Photosystem I. A specific effect on iron-sulfur Center B

Modification of chloroplast membranes with diazonium benzene sulfonate (DABS) leads to a loss of Photosystem I-dependent ferredoxin reduction but not methyl viologen reduction. EPR studies of DABS-modified membranes show no inhibition of P-700⁺ formation at cryogenic temperatures, but iron-sulfur Center A photoreduction is markedly inhibited. Iron-sulfur Center B photoreduction at physiological temperatures in DABS-modified membranes is also markedly inhibited and little Center B can be detected after dark chemical reduction. These results indicate DABS specifically modifies iron-sulfur Center B of the spinach chloroplast Photosystem I electron acceptor complex and that Center B is obligately required for the reduction of Center A at cryogenic temperatures. Possible electron transport pathways at physiological temperatures are also considered.     

Evidence for the involvement of PSI-E subunit in the reduction of ferredoxin by photosystem I.

Of the stroma-accessible proteins of photosystem I (PSI) from Synechocystis sp. PCC 6803, the PSI-C, PSI-D and PSI-E subunits have already been characterized, and the corresponding genes isolated. PCR amplification and cassette mutagenesis were used in this work to delete the psaE gene. PSI particles were isolated from this mutant, which lacks subunit PSI-E, and the direct photoreduction of ferredoxin was investigated by flash absorption spectroscopy. The second order rate constant for reduction of ferredoxin by wild type PSI was estimated to be approximately 10(9) M-1s-1. Relative to the wild type, PSI lacking PSI-E exhibited a rate of ferredoxin reduction decreased by a factor of at least 25. After reassociation of the purified PSI-E polypeptide, the original rate of electron transfer was recovered. When a similar reconstitution was performed with a PSI-E polypeptide from spinach, an intermediate rate of reduction was observed. Membrane labeling of the native PSI with fluorescein isothiocyanate allowed the isolation of a fluorescent PSI-E subunit. Peptide analysis showed that some residues following the N-terminal sequence were labeled and thus probably accessible to the stroma, whereas both N- and C-terminal ends were probably buried in the photosystem I complex. Site-directed mutagenesis based on these observations confirmed that important changes in either of the two terminal sequences of the polypeptide impaired its correct integration in PSI, leading to phenotypes identical to the deleted mutant. Less drastic modifications in the predicted stroma exposed sequences did not impair PSI-E integration, and the ferredoxin photoreduction was not significantly affected. All these results lead us to propose a structural role for PSI-E in the correct organization of the site involved in ferredoxin photoreduction.     

Ferredoxin-NADP+ reductase. Kinetics of electron transfer, transient intermediates, and catalytic activities studied by flash-absorption spectroscopy with isolated photosystem I and ferredoxin

The electron transfer cascade from photosystem I to NADP+ was studied at physiological pH by flash-absorption spectroscopy in a Synechocystis PCC6803 reconstituted system comprised of purified photosystem I, ferredoxin, and ferredoxin-NADP+ reductase. Experiments were conducted with a 34-kDa ferredoxin-NADP+ reductase homologous to the chloroplast enzyme and a 38-kDa N-terminal extended form. Small differences in kinetic and catalytic properties were found for these two forms, although the largest one has a 3-fold decreased affinity for ferredoxin. The dissociation rate of reduced ferredoxin from photosystem I (800 s(-1)) and the redox potential of the first reduction of ferredoxin-NADP+ reductase (-380 mV) were determined. In the absence of NADP+, differential absorption spectra support the existence of a high affinity complex between oxidized ferredoxin and semireduced ferredoxin-NADP+ reductase. An effective rate of 140-170 s(-1) was also measured for the second reduction of ferredoxin-NADP+ reductase, this process having a rate constant similar to that of the first reduction. In the presence of NADP+, the second-order rate constant for the first reduction of ferredoxin-NADP+ reductase was 20% slower than in its absence, in line with the existence of ternary complexes (ferredoxin-NADP+ reductase)-NADP+-ferredoxin. A single catalytic turnover was monitored, with 50% NADP+ being reduced in 8-10 ms using 1.6 microM photosystem I. In conditions of multiple turnover, we determined initial rates of 360-410 electrons per s and per ferredox-in-NADP+ reductase for the reoxidation of 3.5 microM photoreduced ferredoxin. Identical rates were found with photosystem I lacking the PsaE subunit and wild type photosystem I. This suggests that, in contrast with previous proposals, the PsaE subunit is not involved in NADP+ photoreduction.     

Regulation of Cyclic Photophosphorylation during Ferredoxin-Mediated Electron Transport : Effect of DCMU and the NADPH/NADP Ratio

Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O₂ (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP⁺ ratio is varied, the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.     

Two molecular forms of pea ferredoxin in the electron transport chain of chloroplasts

The effects of two molecular forms of water-soluble ferredoxin (Fd I and Fd II) on the kinetics of electron transport in bean chloroplasts (class B) were studied. The light-induced redox transitions of the photosystem I reaction center P700 were measured by the intensity of the EPR signal I produced by P700+. Both forms of ferredoxin, Fd I and Fd II, when added to the chloroplasts in catalytic amounts, stimulate the light-induced electron transfer from P700 to NADP+. Nevertheless, Fd I is a better mediator of the back reactions from NADPH to P700+. This electron transfer pathway is sensitive to the cyclic electron transport inhibitor, antimycin A, and to DCMU inhibitor of electron transport between photosystem II and plastoquinone. It may be concluded that the two molecular forms of ferredoxin, Fd I and Fd II, differ in their ability to catalyze cyclic electron transport in photosystem I. The role of Fd I and Fd II in regulation of electron transport at the acceptor site of photosystem I is discussed.     

The water-water cycle as alternative photon and electron sinks

The water-water cycle in chloroplasts is the photoreduction of dioxygen to water in photosystem I (PS I) by the electrons generated in photosystem II (PS II) from water. In the water-water cycle, the rate of photoreduction of dioxygen in PS I is several orders of magnitude lower than those of the disproportionation of superoxide catalysed by superoxide dismutase, the reduction of hydrogen peroxide to water catalysed by ascorbate peroxidase, and the reduction of the resulting oxidized forms of ascorbate by reduced ferredoxin or catalysed by either dehydroascorbate reductase or monodehydroascorbate reductase. The water-water cycle therefore effectively shortens the lifetimes of photoproduced superoxide and hydrogen peroxide to suppress the production of hydroxyl radicals, their interactions with the target molecules in chloroplasts, and resulting photoinhibition. When leaves are exposed to photon intensities of sunlight in excess of that required to support the fixation of CO2, the intersystem electron carriers are over-reduced, resulting in photoinhibition. Under such conditions, the water-water cycle not only scavenges active oxygens, but also safely dissipates excess photon energy and electrons, in addition to downregulation of PS II and photorespiration. The dual functions of the water-water cycle for protection from photoinhibition under photon excess stress are discussed, along with its functional evolution.     

The kinetics of light-induced changes of C-550, cytochrome b559 and fluorescence yield in chloroplasts at low temperature

The kinetics of the photoreduction of C-550, the photooxidation of cytochrome b₅₅₉ and the fluorescence yield changes during irradiation of chloroplasts at ?196 °C were measured and compared. The photoreduction of C-550 proceeded more rapidly than the photooxidation of cytochrome b₅₅₉ and the fluorescence yield increase followed the cytochrome b₅₅₉ oxidation. These results suggest that fluorescence yield under these conditions indicates the dark reduction of the primary electron donor to Photosystem II, P680⁺, by cytochrome b₅₅₉ rather than the photoreduction of the primary electron acceptor.The photoreduction of C-550 showed little if any temperature dependence over the range of ?196 to ?100 °C. The amount of cytochrome b₅₅₉ photooxidized was sensitive to temperature decreasing from the maximal change at temperatures between ?196 to ?160 °C to no change at ?100 °C. To the extent that the reaction occurred at temperatures between ?160 and ?100 °C the rate was largely independent of temperature. The rate of the fluorescence increase was dependent on temperature over this range being 3–4 times more rapid at ?100 than at ?160 °C. At ?100 °C the light-induced fluorescence increase and the photoreduction of C-550 show similar kinetics. The temperature dependence of the fluorescence induction curve is attributed to the temperature dependence of the dark reduction of P680⁺.The intensity dependence of the photoreduction of C-550 and of the photooxidation of cytochrome b₅₅₉ are linear at low intensities (below 200 μW/cm²) but fall off at higher intensities. The failure of reciprocity in the photoreduction of C-550 at the higher intensities is not explained by the simple model proposed for the Photosystem II reaction centers.