Cyclic electron transport in chloroplasts. The Q-cycle and the site of action of antimycin

Cyclic electron transport systems have been set up in broken chloroplasts, with photochemically reduced ferredoxin or 9,10-anthraquinone-2-sulphonate as cofactor. In good agreement with the literature, only the ferredoxin-catalyzed pathway was found to be inhibited by antimycin; but both pathways were found to have a slow electrogenic reaction, both were inhibited by the cytochrome b-563 oxidation inhibitor 2-heptyl-4-hydroxyquinoline N-oxide (the inhibition being strongest at limiting light intensity), and the two pathways had the same proton/electron stoichiometry at limiting light intensity. It is concluded that a Q-cycle can occur in cyclic electron transport with either cofactor; and therefore that the site of action of antimycin in chloroplasts is not within the Q-cycle, as it is believed to be in mitochondria and bacteria. Instead, a ferredoxin-quinone reductase is proposed as the site of action of antimycin in the ferredoxin-catalyzed cyclic pathway. It is also concluded that the data presented here are consistent with the suggestion that the Q-cycle in photosynthetic electron transport is a facultative one, its degree of engagement depending on competition between the Rieske centre and cytochrome b-563 for reducing equivalents from plastosemiquinone.     

The effect of redox potential on the kinetics of fluorescence induction in pea chloroplasts. II. Sigmoidicity

The shape of the fluorescence induction curve in chloroplasts inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea has been determined at different redox potentials. At ?10 mV a monophasic and sigmoidal curve is seen which is transformed into an exponential curve when the potential is poised at ?150 mV. At this potential, the quencher with high midpoint, Q_H, is reduced but that with low midpoint, Q_L, is oxidized. Thus, a sigmoidal induction is observed during photoreduction of Q_L and Q_H but photoreduction of Q_L proceeds with exponential kinetics. A correlation between the relative proportions of Q_L and Q_H observed in redox titration and the sigmoidicity of induction is also seen upon depletion of Mg²⁺ and after alkalinization to pH 9.5. Several models are discussed to explain the relationship between Photosystem II interactions and Q heterogeneity.     

The electron spin relaxation of the electron acceptors of Photosystem I reaction centre studied by microwave power saturation

Photosystem I particles from spinach were reduced by illumination at 77 K. Under these conditions the one-electron transfer from P-700 resulted in a reduction of only one acceptor molecule of the reaction centre. The EPR signals at g = 2.05, 1.94 and 1.86 were attributed to reduced centre A and the smaller signals at g = 2.07, 1.92 and 1.89 to reduced centre B. Reduction of both centres by dithionite in the dark lead to signals at g = 2.05, 1.99, 1.96, 1.94, 1.92 and 1.89. Thus, the features at g = 2.07 and 1.86 disappeared and new signals at g = 1.99 and 1.96 were observed. From the spectral changes it followed that the iron-sulphur centres A and B interact magnetically. Temperature dependent EPR spectra demonstrated a faster electron spin relaxation of centre A than of centre B.

These conclusions were corroborated using microwave power saturation of the respective EPR signals. The saturation data of the fully reduced centres A and B could not be fitted using the saturation equation for a one-electron spin system. The magnetic interaction between the [4Fe-4S] centres of the electron acceptors A and B resulted in saturation properties which are similar to those of the 2[4Fe-4S] ferredoxin from Clostridium pasteurianum.

For centre X a high proportion of homogeneous broadening of the EPR lines was inferred from the inhomogeneity parameter (b = 1.83). It was, therefore, concluded that centre X is most probably an anion radical of chlorophyll. From the low temperature necessary for observing the EPR signal of centre X followed that the drastic relaxation enhancement has to be attributed to a magnetic interaction of the anion radical with iron.     

The dependence of reaction center and antenna triplets on the redox state of Photosystem I

The formation of chlorophyll triplet states during illumination of Photosystem I reaction center samples depends upon the redox state of P-700, X and ferredoxin Centers A and B. When the reaction centers are in the states P-700⁺A₁XFd_BFd^?_A and P-700 A₁XFd^?_BFd^?_A prior to illumination, we observe electron paramagnetic resonance (EPR) spectra from a triplet species which has zero-field splitting parameters (|D| and |E|) larger than those of either the chlorophyll a or chlorophyll b monomer triplet, and a polarization which results from population of the triplet spin sublevels by an intersystem crossing mechanism. We interpret this triplet as arising from photoexcited chlorophyll antenna species associated with reaction centers in the states P-700⁺Fd^?_A and P-700⁺X^?, respectively, which undergo de-excitation via intersystem crossing. When the reaction centers are in the states P-700A₁XFd^?_BFd^?_A and P-700A₁X^?Fd^?_BFd^?_A prior to illumination, we observe a triplet EPR signal with a polarization which results from population of the triplet spin sublevels by radical pair recombination, and which has a |D| value similar to that of chlorophyll a monomer. We interpret this triplet (the radical pair-polarized triplet) as arising from ³P-700 which has been populated by the process $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{→}{}^3\text{P-700}\text{A}{}_1$. We observe both the radical pair-polarized triplet and the chlorophyll antenna triplet when the reaction centers are in the state P-700 A₁XFd^?_BFd^?_A, presumably because the processes P-700⁺A^?₁X → P-700⁺A₁X^? and $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{X}\text{→}{}^3\text{P-700}\text{A}{}_1\text{X}$ have similar rate constants when Centers A and B are reduced, i.e., the forward electron transfer time from A^?₁ to X is apparently much slower in the redox state P-700 A₁XFd^?_BFd^?_A than it is in state P-700 A₁XFd_BFd_A. The amplitude of the radical pair-polarized triplet EPR signal does not decrease in the presence of a 13.5-G-wide EPR signal centered at g 2.0 which was recorded in the dark prior to triplet measurements in samples previously frozen under intense illumination. This g 2.0 signal, which has been attributed to phototrapped A^?₁ (Heathcote, P., Timofeev, K.N. and Evans, M.C.W. (1979) FEBS Lett. 101, 105–109), corresponds to as many as 12 spins per P-700 and can be photogenerated during freezing without causing any apparent attenuation of the radical pair-polarized triplet amplitude. We conclude that species other than A^?₁ contribute to the g 2.0 signal.     

Photosystem I photochemistry at low temperature. Heterogeneity in pathways for electron transfer to the secondary acceptors and for recombination processes

Electron transport has been studied by flash absorption and EPR spectroscopies at 10–30 K in Photosystem I particles prepared with digitonin under different redox conditions. In the presence of ascorbate, an irreversible charge separation is progressively induced at 10 K between P-700 and iron-sulfur center A by successive laser flashes, up to a maximum which corresponds to about two-thirds of the reaction centers. In these centers, heterogeneity of the rate for center A reduction is also shown. In the other third of reaction centers, the charge separation is reversible and relaxes with a t_1/2 ≈ 120 μs. When the iron-sulfur centers A and B are prereduced, the 120 μs relaxation becomes the dominant process (70–80% of the reaction centers), while a slow component (t_1/2 = 50–400 ms) reflecting the recombination between P-700⁺ and center X⁻ occurs in a minority of reaction centers (10–15%). Flash absorption and EPR experiments show that the partner of P-700⁺ in the 120 μs recombination is neither X nor a chlorophyll but more probably the acceptor A⁻₁ as defined by Bonnerjea and Evans (Bonnerjea, J. and Evans, M.C.W. (1982) FEBS Lett. 148, 313–316). The role of center X in low-temperature electron flow is also discussed.     

The effect of redox potential of the kinetics of fluorescence induction in pea chloroplasts I. Removal of the slow phase

The effect of alteration of redox potential on the kinetics of fluorescence induction in pea chloroplasts has been investigated. Potentiometric titration of the initial (F_i) level of fluorescence recorded upon shutter opening gave a two component curve, with E_m(7) at ?20 mV and ?275 mV, almost, identical to results obtained using continuous low intensity illumination (Horton, P. and Croze, E. (1979) Biochim. Biophys. Acta 545, 188–201). The slow or tail phase of induction observed in the presence of DCMU can be eliminated by poising the redox potential at approx. 0 to +50 mV. At this potential F_i was increased by less than 10% and the higher potential quencher described above was only marginally reduced. The disappearance of the slow phase titrated as an n = 1 component with an E_m(7) of +120 mV. Therefore it seems unlikely that the slow phase of fluorescence induction is due to photoreduction of the ?20 mV quencher. These results are discussed with reference to current ideas concerning heterogeneity on the acceptor side of Photosystem II.     

A study of chemically induced dynamic electron polarization (CIDEP) in Photosystem I of whole algal cells at ambient temperatures

Time-resolved electron paramagnetic resonance (EPR) studies were carried out at room temperature and at 273 K on whole-cell samples of the photosynthetic algae: Anacystis nidulans and Scenedesmus obliquus, the latter being 97% deuterated from the growing medium. These photosynthetic organisms show greatly enhanced EPR signals which result from the generation of nonequilibrium spin populations, a phenomenon known as chemically induced dynamic electron polarization (CIDEP). We report magnetic-field profiles of the early transient signals of Photosystem I which are very similar to those observed at low temperatures. The results suggest that one or more early reduced electron acceptors in Photosystem I are being observed at ambient physiological temperatures.     

Enhancement studies on algae and isolated chloroplasts. Part I. Variability of photosynthetic enhancement in Chlorella phyrenoidosa

Studies of the variability of enhancement in Chlorella pyrenoidosa confirm the existence of two types of variability: a very slow diurnal variation linked to the growth cycle and a much more rapid adaptive response to the immediate incident light conditions (State I–State II transitions). Measurements of the wavelength dependencies and relative contributions of these two types of variability suggest that they may be linked.A close examination of the enhancement signals associated with the State I–State II transition reveals that the transitions can take place in any one of three ways: by a change in Photosystem II efficiency alone, by a change in Photosystem I efficiency alone or by a simultaneous change in the efficiencies of both photosystems.Measurements of the rates of transition between State I, State II and the dark adapted state, Dark, suggest that the behaviour of State II and Dark are normally, but not always, identical. The transitions between the three states were found to be first order. For those samples exhibiting the same behaviour in Dark and State II, the rate of the State I–State II transition was found to be independent of the wavelength of Light II, suggesting that the return from State I to State II is essentially a dark process and that the driving force for the adaptive transition is the over-stimulation of Photosystem I.Finally, a model is proposed, involving an antagonistic control of the quantum yields of photochemistry of the two photosystems, that is capable of explaining the links between the two types of variability, their wavelength dependencies and the shapes of the individual enhancement signals.     

Different kinetics of membrane potential formation in dark-adapted and preilluminated chloroplasts

The effects of varying dark interval on the kinetics of light-induced formation of the membrane potential were studied on individual chloroplasts of Anthoceros with the use of capillary microelectrodes. Illumination of the chloroplast with 1 s light pulse after 3 min dark period induced the photoelectrical response with two peaks of the potential that were located at 20 and 500 ms after the onset of illumination. The position of the second peak was shifted along the time-scale depending on the preceding dark interval. The repeated illumination of the chloroplast with 1 s light pulse after 30 s dark interval induced the electrical response with only one maximum and a monotonous decay of the potential in the light. Distinctions in the electrical responses induced by the first and the second light pulses were eliminated by the addition of 50 μM dicyclohexylcarbodiimide (DCCD). The results show that the photoinduction kinetics of the membrane potential in chloroplasts is affected by functioning of H⁺-ATPase. The delayed peak of the membrane potential in the photoinduction kinetics is interpreted as a consequence of the photoactivated electron transport supported by Photosystem I.     

Redox potential of the non-heme iron complex in bacterial photosynthetic reaction center

In bacterial photosynthetic reaction centers (bRC), the electron is transferred from the special pair (P) via accessory bacteriochlorophyll (B_A), bacteriopheopytin (H_A), the primary quinone (Q_A) to the secondary quinone (Q_B). Although the non-heme iron complex (Fe complex) is located between Q_A and Q_B, it was generally supposed not to be redox-active. Involvement of the Fe complex in electron transfer (ET) was proposed in recent FTIR studies [A. Remy and K. Gerwert, Coupling of light-induced electron transfer to proton uptake in photosynthesis, Nat. Struct. Biol. 10 (2003) 637-644]. However, other FTIR studies resulted in opposite results [J. Breton, Steady-state FTIR spectra of the photoreduction of Q_A and Q_B in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones, Biochemistry 46 (2007) 4459-4465]. In this study, we calculated redox potentials of Q_A/B (E_m(Q_A/B)) and the Fe complex (E_m(Fe)) based on crystal structure of the wild-type bRC (WT-bRC), and we investigated the energetics of the system where the Fe complex is assumed to be involved in the ET. E_m(Fe) in WT-bRC is much less pH-dependent than that in PSII. In WT-bRC, we observed significant coupling of ET with Glu-L212 protonation upon oxidation of the Fe complex and a dramatic E_m(Fe) downshift by 230 mV upon formation of Q_A⁻ (but not Q_B⁻) due to the absence of proton uptake of Glu-L212. Changes in net charges of the His ligands of the Fe complex appear to be the nature of the redox event if we assume the involvement of the Fe complex in the ET.     

Phosphorylation of chloroplast thylakoids decreases the maximum capacity of photosystem-II electron transfer

Phosphorylation of chloroplast thylakoid polypeptides by the light-activated protein kinase was found to decrease the light-saturated rate of whole chain and Photosystem-II electron transport. This decrease in electron-transport capacity was reversible and was found to correlate with the phosphorylation-induced decrease in chlorophyll fluorescence.     

The influence of anions on oxygen evolution by isolated spinach chloroplasts

A study has been made of the onset of chloride deprivation on the oxygen-evolving characteristics of isolated spinach chloroplasts. Using a modulated oxygen electrode it is found that the type of inhibition depends on the anion replacing chloride in the bathing medium. With nitrate a large increase in phase lag accompanies a relatively small inhibition which can be shown to be consistent with a decrease in the rate constant of the reaction which limits the rate of electron transport between water and Photosystem II. With sulphate there is a very small phase change but a larger inhibition which suggests that replacing chloride with sulphate in an electron-transport chain shuts off that chain. With acetate there is a moderate increase in phase lag and the largest inhibitory effect. The phase-lag increase suggests that acetate is affecting the same chloride-sensitive site as nitrate. However, the inhibition cannot be explained by this effect alone and points to the existence of a second chloride-sensitive site. Of the four forward reactions associated with the Kok model of oxygen evolution (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) only S1₃ → S₀ is slowed down when chloride is replaced by nitrate. This reaction is not slowed down by replacing chloride with sulphate.     

Cytochrome function in the cyclic electron transport pathway of chloroplasts

Flash excitation of isolated intact chloroplasts promoted absorbance transients corresponding to the electrochromic effect (P-518) and the α-bands of cytochrome b₆ and cytochrome f. Under conditions supporting coupled cyclic electron flow, the oxidation of cytochrome b₆ and the reduction of cytochrome f had relaxation half-times of 15 and 17 ms, respectively. Optimal poising of cyclic electron flow, achieved by addition of 0.1 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea, increased phosphorylation of endogenous ADP and prolonged these relaxation times. The presence of NH₄Cl, or monensin plus NaCl, decreased the half-times for cytochrome relaxation to approximately 2 ms. Uncouplers also revealed the presence of a slow rise component in the electrochromic absorption shift, with formation half-time of about 2 ms. The inhibitors of cyclic phosphorylation antimycin and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone abolished the slow rise in the electrochromic shift and prolonged the uncoupled relaxation times of cytochromes b₆ and f by factors of ten or more.These observations indicate that cytochrome b₆, plastoquinone and cytochrome f participate in a coupled electron transport process responsible for cyclic phosphorylation in intact chloroplasts. Estimations of cyclic phosphorylation rates from 40 to 120 μmol ATP/mg chlorophyll per h suggest that this process can provide a substantial fraction of the ATP needed for CO₂ fixation.     

Regulation of photosynthetic electron transport and photophosphorylation in intact chloroplasts and leaves of Spinacia oleracea L.

Oxygen ist reduced by the electron transport chain of chloroplasts during CO₂ reduction. The rate of electron flow to oxygen is low. Since antimycin A inhibited CO₂-dependent oxygen evolution, it is concluded that cyclic photophosphorylation contributes ATP to photosynthesis in chloroplasts which cannot satisfy the ATP requirement of CO₂ reduction by electron flow to NADP and to oxygen. Inhibition of photosynthesis by antimycin A was more significant at high than at low light intensities suggesting that cyclic photophosphorylation contributes to photosynthesis particularly at high intensities. Cyclic electron flow in intact chloroplasts is under the control of electron acceptors. At low light intensities or under far-red illumination it is decreased by substrates which accept electrons from photosystem I such as oxaloacetate, nitrite or oxygen. Obviously, the cyclic electron transport pathway is sensitive to electron drainage. In the absence of electron acceptors, cyclic electron flow is supported by far-red illumination and inhibited by red light. The inhibition by light exciting photosystem II demonstrated that the cyclic electron transport pathway is accessible to electrons from photosystem II. Inhibition can be relieved by oxygen which appears to prevent over-reduction of electron carriers of the cyclic pathway and thus has an important regulatory function. The data show that cyclic electron transport is under delicate redox control. Inhibition is caused both by excessive oxidation and by over-reduction of electron carriers of the pathway.     

Extremely fast photoelectric signals from suspensions of broken chloroplasts and of isolated chromatophores

This paper concerns transient photoelectric signals that are observed when a suspension of photosynthetic vesicles is illuminated with flashing light of nonsaturating energy using a pair of electrodes positioned at different depths along the path of the exciting light. With chloroplasts we observed two different types of signals. One rose extremely fast, the rise time (10–90%) was less than 200 ps under excitation with a single pulse from a mode-locked ruby laser, while the other rose more slowly (typically 10 μs). These signals displayed several different properties such as their polarity, kinetics, apparent source impedance, and sensitivity to structural integrity of the chloroplast lamellar system. Experimentally, signal ‘Fast’ could only be induced by very short light pulses (shorter than approx. 60 ns), whereas signal ‘Slow’ appeared only under longer excitation. The detection of signal Fast required special instrumentation, particularly fast preamplifiers with low input capacitance. Our results support the more recent idea that signal Slow did not reflect the primary transmembrane charge separation, as postulated in the earlier literature, but rather lateral movement of charge carriers along the intact lamellar system of chloroplasts. On the other hand, signal Fast may reflect the primary outwardly directed electron transfer across the thylakoid membrane. Its polarity, however, was opposite to that previously postulated to appear in response to this event. For comparison we also studied photoelectric effects in a suspension of structurally more homogeneous chromatophores from Rhodopseudomonas sphaeroides. These vesicles displayed only a signal of the same polarity and similar kinetics as signal Fast from chloroplasts. When the secondary quinone electron acceptor (Q) was chemically reduced, the decay time was shortened from approx. 30 ns to approx. 10 ns. The acceleration to approx. 10 ns is known for the rapid, supposedly transmembrane back-reaction of the primary charge separation. Therefore, we conclude that the electrodes monitor the primary charge separation. There is still the unresolved problem with the polarity of the electric signal which we attribute to an as yet unidentified property of the pick-up system. Because its signal-to-noise ratio under extremely high time resolution is superior to that obtained in flash spectroscopy, signal Fast represents a very good means to measure the spatial separation between the very primary electron carriers in the photosynthetic reaction center.     

Equilibrium and kinetic measurements of the redox potentials of cytochromes c2 in vitro and in vivo

The equilibrium oxidation-reduction mipoint potential (E_m) of isolated Rhodopseudomonas sphaeroides cytochrome c₂ exhibits a pH-dependent behavior which can be ascribed to a pK on the oxidized form at pH 8.0 (Pettigrew et al. (1975) Biochim. Biophys. Acta 430, 197–208). However, as with mammalian cytochrome c (Brandt, K.G., Parks, P.C., Czerlinski, G.H. and Hess, G.P. (1966) J. Biol. Chem. 241, 4180–4185) this pK can more properly be attributed to the combination of a pK beyond pH 11, and a slow conformational change of the ferricytochrome. This has been demonstrated by resolving the E_m of cytochrome c₂ before and after the conformational change. The E_m of the unaltered form is essentially pH independent between pH 7 and 11.5, and the lower equilibrium E_m is due solely to the conformational change. In vivo the conformational change is prevented by the binding of the cytochrome c₂ to the photochemical reaction center, and the cytochrome exhibits an essentially pH-independent E_m from pH 5 to 11. The alkaline transition thus has little physiological significance, and it is unlikely that the redox reactions of cytochrome c₂ in vivo involve protons.     

ESR characteristics of Photosystem I in deuterium oxide: Further evidence that electron acceptor A1 is a quinone

The appearance of ESR signals from Photosystem I (PS I) electron acceptors A₁ and A₀ in water or deuterium oxide suspension was followed using a low-temperature photoaccumulation technique. In deuterated samples the A₁ signal was narrowed by a factor of 0.66 compared with the control. This effect was fully reversible upon resuspension of treated samples in H₂O. The narrow ESR signal from deuterated A₁ had similar power saturation characteristics to the normal signal; however, a signal from a second component resolved by deuteration was saturated at higher microwave powers than the control. The power saturation behaviour of A⁻₁ in un-modified reaction centres indicated that it is an anionic semiquinone in a ‘protic’ environment. Deuteration reversibly modified the relative extents of reduction of iron sulphur electron acceptors A and B such that centre B became the more stable electron acceptor. The g-value and line-width of iron sulphur centre X was not modified by deuteration although it appeared to become more efficiently reduced. These results are discussed in the light of current evidence from optical, electron spin polarisation and extraction experiments that suggest that A₁ is a quinone, probably vitamin K-1.     

Heterogeneity of system II secondary electron acceptors in Tris-washed chloroplasts

Tris-washed chloroplasts were submitted to saturating short flashes, and then rapidly mixed with dichlorophenyldimethylurea (DCMU). The amount of singly reduced secondary acceptor was estimated from the DCMU-induced increase in fluorescence, caused by the reverse electron flow from secondary to primary acceptor. The back-transfer from the singly reduced secondary acceptor to the primary acceptor Q induced by DCMU addition affects only a part (60%) of the variable fluorescence (ΔF_max). As previously shown, the quenchers involved in this phenomenon, ‘B-type’ quenchers, are different from those controlling the complementary part of the fluorescence, the non-B-type. In this report, we show that at pH 8.5 in the B-type systems, there exist two kinds of secondary electron acceptors: B, a two-electron acceptor, the corresponding Q accounting for 40% of the variable fluorescence; B′, a one-electron acceptor, the corresponding Q accounting for 20% of the variable fluorescence. The lifetimes of B^? and B′^? in the absence of DCMU are 40 and 1 s, respectively. The primary acceptors of the B and B′ systems can be considered as corresponding to the Q₁s defined previously (Joliot, P. and Joliot, A. (1981) in Proceedings of the 5th International Congress on Photosynthesis (Akoynoglou, G., ed.), pp. 885–899, Balaban International Science Services, Philadelphia). The B′ centers seems to be equivalent to the Q_β centers as defined by other workers (Van Gorkom, H.J., Thielen, A.P.G.M. and Gorren, A.C.F. (1982) in The Function of Quinones in Energy Conserving Systems (Trumpower, B.L., ed.), Academic Press, New York, in the press).     

Evidence that the low-potential (−700 mV) electron acceptor (X) in Photosystem I has two iron-sulphur centres

The Photosystem I reaction centre contains two groups of iron-sulphur centres: Fe-S_A and Fe-S_B with redox potentials between ?510 and ?590 mV, and Fe-S_X with redox potential about ?700 mV. Spin quantitation (Heathcote, P., Williams-Smith, D.L. and Evans, M.C.W. (1978) Biochem. J. 170, 373–378) and Mössbauer spectroscopy (Evans, E.H., Dickson, D.P.E., Johnson, C.E., Rush, J.D. and Evans, M.C.W. (1981) Eur. J. Biochem. 118, 81–84) did not show unequivocally whether Fe-S_X has one or two centres. Experiments are described which support the proposal that Fe-S_X has two centres. Fe-S_X can be photoreduced irreversibly by 210 K illumination of dithionite-reduced samples or reversibly by 7.5 K illumination of these samples. The amplitude of the Fe-S_X signal reversibly induced by illumination at 7.5 K is never more than 50% of the amplitude of the signal when Fe-S_X is prereduced by room temperature illumination or by 210 K illumination. Approx. half of the Fe-S_X is rapidly reduced by 210 K illumination, the remainder more slowly. The extent of reversible Fe-S_X reduction and P-700 photooxidation is little affected by the fast reduction of about half of the Fe-S_X. Subsequent reduction of the remaining Fe-S_X is paralleled by loss of the reversible photoreaction.