Studies on well-coupled Photosystem I-enriched subchloroplast vesicles — characteristics and reinterpretation of single-turnover cyclic electron transfer

The contributions of ferredoxin, P-700, plastocyanin and the cytochromes c-554, and b-563 to single-turnover electron transfer in Photosystem (PS) I-enriched subchloroplast vesicles were deconvoluted by fitting the literature-derived spectra of these components to the observed absorption data at a series of wavelengths, according to a linear least-squares method. The obtained corresponding residuals showed that the applied component spectra were satisfactory. The deconvoluted signals of cytochromes c-554 and b-563 differed in some cases significantly from the classical dual-wavelength signals recording at 554–545 nm and 563–575 (or −572) nm, due to interference from other electron-transferring components. KCN, DNP-INT (2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether), DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzo-quinone) and antimycin A all inhibited electron transfer, although antimycin and DBMIB inhibited only after a few turnovers of the cytochrome bf complex. Fast flash-induced reduction of cytochrome b-563 exclusively reflected oxidant-induced reduction. Fast electron flow from cytochrome c-554 to plastocyanin and P-700 resulted in an apparent rereduction of cytochrome c-554 that was slower than the reduction of cytochrome b-563. Model simulations indicate that under highly oxidizing conditions for the Rieske FeS centre and reducing conditions for cytochrome b-563, the semiquinone at the Q_z site cannot only reduce cytochrome b-563, but can also oxidize cytochrome b-563 and reduce the Rieske FeS centre. The effect of 10 μM gramicidin D was evaluated in order to determine the contributions by electrochromic absorption changes around 518 nm. Gramicidin left electron transfer, monitored in the 550–600 nm range, unchanged. The gramicidin-sensitive (membrane potential-associated) signal at 518 nm differed from the signals recorded in the absence of gramicidin at 518 nm or 518–545 nm, due to spectral interference from electron-transferring components in the latter signals. KCN, DBMIB and antimycin A affected both the fast and slow components of the electrochromic signal, but did not proportionally affect the initial electron transfer from P-700 to ferredoxin (charge separation in PS I). Not only the slow (10–100 ms) component of the 518 nm absorption change, but also part of the fast (less than 1 ms) component appears to minitor electrogenic events in the cytochrome bf complex.     

The effects of quinone analogues on cytochrome b6 reduction and oxidation in a reconstituted system

The reconstituted system containing Photosystem I, plastocyanin and the cytochrome b6-f complex is used to study the effects of various quinone analogues on the redox behavior of cytochrome b6. The effects of DBMIB, DNP-INT and HQNO are compared in an attempt to discern the modes of action of these quinone analogues. Both DBMIB and DNP-INT are potent inhibitors of the plastocyanin reductase activity of the isolated cytochrome complex. However, while DBMIB abolished the oxidant-induced reduction of cytochrome b6, DNP-INT only inhibited about 25% of the net reduction. On the other hand, HQNO does not show any significant inhibition of plastocyanin reductase activity of the isolated cytochrome complex at concentrations up to 20 microM. An enhancement of the net amount of cytochrome b6 reduced is observed in the presence of HQNO. Both DNP-INT and HQNO inhibited the dark oxidation rate of cytochrome b6. The possible identity of the oxidant for cytochrome b6 is discussed. Plastoquinone is concluded to be the most likely candidate. DNP-INT is concluded to have at least two sites of inhibition in the cytochrome complex. The implications of these findings on quinone functions in the cytochrome b6-f complex are discussed.     

Relationship between P700 turnover (m second) and NADP reduction as a function of ferredoxin concentration in isolated broken chloroplasts

Steady-state electron flux through P₇₀₀ (t $\text{1}\text{2}$ 20 msec) and concomitant rate of NADP reduction have been measured under weak actinic illumination as a function of concentration of ferredoxin added to broken chloroplasts isolated from peas. At suboptimal concentrations of ferredoxin this P₇₀₀ is not sufficient to account for the NADP reduction. At high concentrations ferredoxin inhibits the rate of NADP reduction without affecting the P₇₀₀ flux under short wavelength illumination. Under far red illumination P₇₀₀ flux is also inhibited by ferredoxin at high concentrations. Addition of 5 mM Mg⁺⁺ increases the rate of NADP reduction at all concentrations of ferredoxin under both kinds of illumination, while P₇₀₀ flux is inhibited under short wavelength illumination and remains unchanged under far red illumination. The results indicate that the observed (20 msec) P₇₀₀ is not involved in NADP reduction.     

Differential inhibition by plastoquinone analogues of photoreduction of cytochrome b-559 in chloroplasts

The high-potential form of cytochrome b-559 (b-559 HP) is closely linked to the oxygenic photosystem (photosystem II) but its relation to other redox components of the photosynthetic apparatus, including plastoquinone, is still obscure. We investigated the photoreduction of cytochrome b-559 HP by isolated chloroplasts in the presence of 3 antagonists of plastoquinone, of which, DBMIB (dibromothymoquinone) and DNP-INT (dinitrophenyl ether of iodonitrothymol) are known to inhibit the oxidation of the plastoquinone pool (PQ) by the FeS-cytochrome ƒ/b₆ complex and one, UHDBT (5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole) is known to inhibit the reduction of PQ by Q_B.Q_B is a protein-bound plastoquinone that serves as a two-electron gate for the reduction of PQ. We found that DBMIB and DNP-INT did not inhibit but low concentrations of UHDBT severely inhibited the photoreduction of cytochrome b-559 HP. These results suggest that the electron donor for the reduction of cytochrome b-559 HP was either Q_B or a portion of the PQ pool that was oxidized by a new pathway free of binding sites for DBMIB and DNP-INT.     

Three Substrate Binding Sites on Spinach Ferredoxin:NADP+ Oxidoreductase. Studies with Selectively Acting Inhibitors

The effects of phenylmercuric acetate (PMA) and apoferredoxin (apoFd) on the diaphorase activity of spinach ferredoxin:NADP⁺ oxidoreductase (FNR) in the presence of dibromothymoquinone (DBMIB) or cytochrome c (Cyt c) were studied. PMA inhibited effectively (I₅₀ = < 5 M) ferredoxin-dependent Cyt c reduction but did not affect evidently the enzyme activity in the presence of DBMIB as an electron acceptor. ApoFd caused also inhibition of Cyt c reduction but slightly stimulated, like ferredoxin, DBMIB reduction. We confirm a hypothesis according to which three binding sites for substrates [NADP(H), Fd-Cyt c, quinone/dichlorophenol indophenol] occur within the molecule of isolated FNR.     

Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

A cell-free preparation of heterocysts from Anabaena variabilis showed high nitrogenase activities with several physiological electron donors, dependent on addition of an ATP-generating system. Light-induced acetylene reduction with the artificial electron donor to photosystem I, diaminodurol, exhibited the same light saturation as with hydrogen as donor. Inhibitors of electron flow through plastoquinone affected light-induced, hydrogen- or NADH-dependent nitrogenase activity in a similar way. Several uncoupling agents were without effect, indicating that energized membranes are not a prerequisite for nitrogen fixation. We conclude that NADH or hydrogen deliver electrons to nitrogenase via photosystem I and ferredoxin, feeding in at the plastoquinone site.In the light, addition of NADP induced a lag in H₂- or NADH-supported acetylene reduction apparently by competing with nitrogenase for electrons at the reducing side of photosystem I. Time reversal of this inibition reflects a regulation of photosystem I-dependent nitrogenase activity by the NADPH/NADP ratio in the cell. This was directly demonstrated by differently adjusted NADPH/NADP ratios.NADPH donates electrons to nitrogenase in the dark and in the light, the light reaction being DBMIB-sensitive. NADPH-supported acetylene reduction was inhibited by NADP. This inhibition was not reversed with time, pointing to an involvement of ferredoxin: NADP oxidoreductase (EC 1.18.1.2) in this pathway. Apparently, in the dark, this enzyme is able to directly reduce ferredoxin, whereas in the light electrons from NADPH first have to pass through photosystem I before reducing ferredoxin, hence nitrogenase.Intermediates of glycolysis, like glucose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate supported nitrogenase activity in the dark, each with catalytic amounts of both NAD and NADP as equally effective cofactors.We conclude that in heterocysts electrons for nitrogen fixation are essentially supplied by dark reactions, mainly by glycolysis. NADH (and hydrogen) contribute electrons via photosystem I in the light, whereas the NADPH/NADP ratio regulates linear and cyclic electron flow at the reducing side of photosystem I to provide a ratio of ATP/electrons most effective for nitrogenase.Abbvreviations ATCC American Type Culture Collection - Diaminodurol (DAD) 2,3,5,6-tetramethyl-p-phenylenediamine dihydrochloride - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DNP-INT 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol - E Einstein (mol photons) - FNR ferredoxin - NADP oxidoreductase (EC 1.18.1.2) - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Metronidazole 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole     

Flash photolysis-electron spin resonance studies of photosystem I. A fast reduction of component of P-700+.

A 300 mus decay component of ESR Signal I (P-700+) in chloroplasts is observed following a 10 mus actinic xenon flash. This transient is inhibited by treatments which block electron transfer from Photosystem II to Photosystem I (e.g. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), KCN and HgCl2). The fast transient reduction of P-700+ can be restored in the case of DCMU or DBMIB inhibition by addition of an electron donor couple (2,6-dichlorophenol indophenol (Cl2Ind)/ascorbate) which supplies electrons to cytochrome f. However, this donor couple is inefficient in restoring electron transport in chloroplasts which have been inhibited with the plastocyanin inactivators, KCN and HgCl2. Oxidation-reduction measurements reveal that the fast P-700+ reduction component reflects electron transfer from a component with Em = 375 +/- 10 mV (pH = 7.5). These data suggest the assignment of the 300-mus decay kinetics to electron transfer from cytochrome f (Fe2+) to P-700+, thus confirming the recent observations of Haehnel et al. (Z. Naturforsch. 26b, 1171-1174 (1971)).     

Oxygenic photoreduction of ferredoxin independently of the membrane-bound iron-sulfur centers of photosystem I

An investigation of the photoreduction of soluble ferredoxin and membrane-bound Fe-S centers of chloroplasts yielded results that are incompatible with some basic postulates of the now prevalent concept of photosynthetic electron transport (the “Z scheme”). In the Z scheme, plastquinone serves as an essential link in a linear electron transport chain from water via photosystem II to photosystem I and thence to the bound Fe-S centers, soluble ferredoxin and NADP⁺. In this formulation the oxygenic photoreduction of ferredoxin and of the Fe-S centers should have the same sensitivity to the plastoquinone inhibitors, dibromothymoquinone (DBMIB) and dinitrophenol ether of iodonitrothymol (DNP-INT). We found that the photoreduction of ferredoxin and the Fe-S centers exhibited differential sensitivity to these inhibitors. Ferredoxin was fully photoreduced by water at inhibitor concentrations that abolished the photoreduction of the Fe-S centers. These findings suggest that the oxygenic photoreduction of ferredoxin does not involve the participation of the Fe-S centers or other components of photosystem I. Only when an artificial, direct donor to photosystem I is used is the reduction of ferredoxin invariably preceded by the reduction of the Fe-S centers.     

Effects of inhibitors on the activity of the cytochrome b(6)f complex: evidence for the existence of two binding pockets in the lumenal site.

The effects of two inhibitors of electron transfer in the cytochrome b(6)f complex have been studied in whole cells of Chlamydomonas reinhardtii. DNP-INT affected equally the two steps of the concerted oxidation of plastoquinol at the Q(o) site; it decreased the rates of both cytochrome f reduction and cytochrome b(6) turnover, without affecting the amplitude of their redox signals. On the contrary, DBMIB inhibited only the rate of cytochrome f reduction while reducing, at the same time, the amplitude of cytochrome b(6) signals. The accessibility of DNP-INT to the Q(o) site was unaffected by preillumination, while that of DBMIB was greatly enhanced, even after a single turnover of the cytochrome b(6)f complex. Similar results were obtained with a mutant strain (FUD2) where the Q(o) site has an affinity for plastoquinol that is diminished by a factor of approximately 50 [Finazzi, G., et al. (1997) Biochemistry 36, 2867-2874]. However, the binding of the two inhibitors was differentially influenced by the mutation: a factor of approximately 250 was calculated for DNP-INT and a factor of only approximately 5 for DBMIB. This suggests that they bind within the Q(o) site in two distinct pockets, which are differentially involved in the process of quinol oxidation, in agreement with a recent model where two distinct positions for the reduced and semireduced quinones are considered [Crofts, A. R., and Berry, E. A. (1998) Curr. Opin. Struct. Biol. 8, 501-509].     

Light-dependent development of photosynthetic competence in Scenedesmus mutant No. 8.

The heterotrophically grown, P-700-free mutant No. 8 of Scenedesmus obliquus is unable to carry out photosynthesis. Yet, chloroplast particles isolated from the alga reduced ferricyanide. They also reduced methyl viologen in the presence of the artificial donor reduced 2,6-dichlorophenol indophenol with a low yield but an appreciable saturation rate. NADP reduction or P-700 turn-over could not be detected. When grown mixotrophically, the mutant showed increasing P-700 activity with a concomitant increase in the rate of photosynthesis. Both activities were lost again when the algae were returned to darkness. Isolated chloroplast particles showed a good P-700 turn-over and reasonable rates of NADP reduction. The data suggest that the mutation occurred at a site preceding the formation of the pigment. The results on the photochemical activities are discussed in the light of reports concerning the involvement of P-700 in linear electron transport.     

Uncouplers enhance photosynthetic electron transport from water to NADP+ in the presence of plastoquinone inhibitors

Uncouplers have been previously observed to relieve appreciably the inhibition of photosynthetic electron transport from water to NADP⁺ by the plastoquinone analogues, dibromothymoquinone (DBMIB) and dinitrophenyl ether of iodonitrothymol (DNP-INT). These results were now extended by demonstrating that the reversal by uncouplers of DBMIB and DNP-INT inhibition occurred under conditions when the uncouplers did not stimulate or inhibit NADP⁺ reduction in control treatments without the plastoquinone analogues. Since effects of uncouplers on photosynthetic electron transport depend on external pH, we determined for each of the four uncouplers, gramicidin, nigericin, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and SF 6847 (a ditertiary phenol derivative) its effect on oxygenic electron transport (H₂O to NADP⁺) over a range of external pH from 6.7 to 8.7. The effect of each uncoupler on counteracting the inhibition of DBMIB and DNP-INT was then measured at its crossover external pH at which the uncoupler had little or no effect on electron transport in the uninhibited controls. Under these controlled conditions, uncouplers increased the rate of plastoquinone-inhibited electron transport, in some cases by almost 300%. To explain these results, a role for plastoquinone in processing protons released by the oxidation of water is postulated.     

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus.

A soybean flour-induced, soluble cytochrome P-450 (P-450soy) was purified 130-fold to homogeneity from Streptomyces griseus. Native cytochrome P-450soy is a single polypeptide, with a molecular weight of 47,500, in association with one ferriprotoporphyrin IX prosthetic group. Oxidized P-450soy exhibited visible absorption maxima at 394, 514, and 646 nm, characteristic of a high-spin cytochrome P-450. The CO-reduced difference spectrum of P-450soy had a Soret maximum at 448 nm. When reconstituted with spinach ferredoxin and spinach ferredoxin:NADP+ oxidoreductase, purified cytochrome P-450soy catalyzed the NADPH-dependent oxidation of the xenobiotic substrates precocene II and 7-ethoxycoumarin. In vitro proteolysis of cytochrome P-450soy generated a stable and catalytically active cytochrome P-450, designated P-450soy delta.     

Generation of superoxide anion and hydrogen peroxide during redox cycling of 5-(4-nitrophenyl)-penta-2,4-dienal by mammalian microsomes and enzymes

5-(4-Nitrophenyl)penta-2,4-dienal (NPPD) stimulated NADPH-supported oxygen consumption by rat liver microsomes in a concentration-dependent manner. The NPPD stimulation of O2 uptake was not inhibited by metyrapone and was decreased in the presence of NADP+ and p-hydroxymercuribenzoate. These observations suggest that the NPPD initial reduction step is mediated by NADPH-cytochrome P-450 reductase and not by cytochrome P-450. Spin-trapping studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the formation of superoxide anion upon incubation of NPPD, NADPH, DMPO and rat liver microsomes. Hydrogen peroxide generation was also detected in these incubations, thus confirming redox cycling of NPPD under aerobic conditions. NPPD stimulated oxygen consumption, superoxide anion formation and hydrogen peroxide generation by rat kidney, testes and brain microsomes. Other enzymes capable of nitroreduction (NADH dehydrogenase, xanthine oxidase, glutathione reductase, and NADP+ ferredoxin oxidoreductase) were also found to stimulate redox cycling of NPPD. The ability of NPPD to induce superoxide anion and hydrogen peroxide formation might play a role in its reported mutagenicity.     

Effects of dibromothymoquinone on oxidation-reduction reactions and the midpoint potential of the Rieske iron-sulfur center in photosynthetic electron transport of Synechococcus Sp.

The effects of 2,5-dibromo-3-methyl-p-benzoquinone (DBMIB) on the reduction kinetics of flash-oxidized P-700 and cytochrome c-553 were studied in the thermophilic cyanobacterium Synechococcus sp. (1) The reduction kinetics of P-700 showed two exponential phases with half-times of 0.2 and 2 ms at the recording time used (Nanba, M. and Katoh, S. (1983) Biochim. Biophys. Acta 725, 272–279). DBMIB strongly slowed down the 2 ms reduction phase but not the 0.2 ms phase. (2) The content of an electron donor which transfers its electrons to P-700 with the half time of 0.2 ms was estimated to be comparable to that of cytochrome f. (3) The magnitudes of the 0.2 ms reduction phase and cytochrome c-553 oxidation decreased as the flash interval was shortened below 2 s in the poisoned cells. Assuming a rapid equilibrium of electrons in the electron donor pool of Photosystem I, the midpoint potential of the 0.2 ms donor was estimated as 280 mV by comparing its percent reduction with that of cytochrome c-553 at three different flash intervals. (4) A similar value was obtained for the midpoint potential of the 0.2 ms donor in the cells in which the plastoquinone pool had been oxidized by dark starvation. It is concluded that the 0.2 ms reduction phase of P-700 is due to the electron donation from the Rieske iron-sulfur center and that DBMIB inhibits strongly but incompletely the reduction of the iron-sulfur center with electrons from the plastoquinone pool, whereas the inhibitor has no effect on the midpoint potential and Photosystem-I-dependent oxidation of the iron-sulfur center.     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl(2), an inhibitor of cytochrome b(6)f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b(6)f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

Participation of ferredoxin in oxygen reduction in a photosynthetic electron-transport chain

Oxygen reduction in a photosynthetic electron-transport chain (PETC) was studied in isolated pea thylakoids in the presence of either ferredoxin, or ferredoxin + NADP+, or cytochrome c. The contribution of the electron flow through ferredoxin to the total oxygen reduction was evaluated by comparing the rate of oxygen reduction and the rate of oxidation of reduced ferredoxin in the light. It was found that at ferredoxin concentrations optimal for NADP+ reduction, 30-50% of electrons transferred to oxygen went through ferredoxin both in the absence and presence of NADP+. However, the absolute rate of oxygen reduction by membrane components of PETC in the presence of NADP+ was 3-4 times less than that in the presence of ferredoxin alone and close to the rate of oxygen reduction in the presence of cytochrome c. It was assumed that a Photosystem I component, whose role in this process depends on the rate of electron outflow from terminal acceptors of this photosystem, participates in oxygen reduction, and this component is phylloquinone.     

Glycolytic Flux and Hexokinase Activities in Anoxic Maize Root Tips Acclimated by Hypoxic Pretreatment

In vitro cyclic electron transport around PSI was studied in thylakoids isolated from barley (Hordeum vulgare L.). Redox poising was obtained by using anaerobic conditions, preillumination, and the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Postillumination rates of P700+ re-reduction of 1 to 5 electrons s-1 were observed, depending on the conditions. The thylakoids supported two parallel paths of cyclic electron transport that were distinguishable by differences in antimycin sensitivity, saturation characteristics, and substrate specificity. The pathway most sensitive to antimycin was not saturated at ferredoxin concentrations up to 50 [mu]M, whereas the more insensitive pathway was saturated at 5 [mu]M ferredoxin. At the lower concentration of reduced ferredoxin, the antimycin-sensitive rate of P700+ re-reduction was lower than the antimycin-insensitive rate. The lower range of reduced ferredoxin concentrations are closer to in vivo conditions. Flavodoxin is shown to mediate cyclic electron transport. Flavodoxin was less efficient in mediating the antimycin-sensitive pathway but mediated the antimycin-insensitive pathway as efficiently as ferredoxin. Antibodies raised against ferredoxin:NADP+ oxidoreductase had no effect on either pathway for re-reduction of P700+. However, the ferredoxin: NADP+ oxidoreductase inhibitor 2[prime]-monophosphoadenosine-5[prime]-diphosphoribose was able to inhibit the antimycin-sensitive as well as the antimycin-insensitive pathway.     

Xenobiotic oxidation by cytochrome P-450-enriched extracts of Streptomyces griseus

Crude extracts of Streptomyces griseus grown on soybean flour-enriched medium contain high levels of cytochrome P-450. The cytochrome P-450-enriched fractions, obtained by ammonium sulfate fractionation (30-50% saturation), catalyze the NADPH-dependent oxidation of a variety of xenobiotics when complemented with both spinach ferredoxin:NADP+ oxidoreductase and spinach ferredoxin. Reactions observed are aromatic, benzylic and alicyclic hydroxylations, O-dealkylation, non-aromatic double bond epoxidation, N-oxidation and N-acetylation.     

Chemical modification of carboxyl groups on spinach ferredoxin alters its ability to interact with ferredoxin:NADP reductase

Treatment of spinach ferredoxin with glycine ethyl ester in the presence of a water soluble carbodiimide resulted in the modification of 3-4 carboxyl groups and decreased the ability of ferredoxin to participate in NADP photoreduction by chloroplast membranes by about 80%. The ability of the modified ferredoxin to receive electrons from the reducing side of Photosystem I was relatively unaffected. These findings suggest that the modified ferredoxin is unable to interact with ferredoxin:NADP reductase. This has been verified by demonstration that the modified ferredoxin fails to produce difference spectra typical of a ferredoxin-ferredoxin:NADP reductase complex when added to ferredoxin:NADP reductase.     

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).