The carrier of photosynthetically generated reducing power is the iron-sulfur protein ferredoxin, which provides directly, or via NADP+, reducing equivalents needed for CO2 assimilation and other metabolic reactions in the cell. It is now widely held that, in oxygenic photosynthesis, the generation of reduced ferredoxin-NADP+ requires the collaboration in series of two photosystems: photosystem II (PSII), which energizes electrons to an intermediate reducing potential and transfers them to photosystem I (PSI), which in turn is solely competent to energize electrons to the strong reducing potential required for the reduction of ferredoxin-NADP+ (the Z scheme). This investigation tested the premise of an alternative scheme, which envisions that PSII, without the involvement of PSI, is also capable of photoreducing ferredoxin-NADP+. We report here unexpected findings consistent with the alternative scheme. Isolated PSII reaction centers (completely free of PSI components), when supplemented with ferredoxin, ferredoxin-NADP+ oxidoreductase, and a PSII electron donor,1,5-diphenylcarbazide, gave a significant photoreduction of NADP+. A striking feature of this electron transfer from a PSII donor to the perceived terminal acceptor of PSI was its total dependence on catalytic quantities of plastocyanin, a copper-containing electron-transport protein hitherto known only as an electron donor to PSI.

Recent work in this and other laboratories has demonstrated that, contrary to the favored Z scheme hypothesis, photosystem II (PS II) can photoinduce electron transfer from water to NADP+, without the participation of photosystem I (PS I). One proposed explanation for this conflict between hypothesis and observation was that PS II can reduce NADP+ but only at high light intensities. We report here findings at variance with this proposal. A PS II preparation made from spinach chloroplasts by the two‐phase aqueous polymer partition method photoreduced NADP+ without the involvement of PS I, at varying light intensities ranging from limiting to saturating.

Although cytochrome b -559 has long been known as a membrane-bound redox component closely linked to the reaction center of the oxygen-generating photosystem (PSII), its role in photosynthesis has remained obscure. This paper reports evidence and outlines a hypothesis in support of a “ b -559 cycle”—i.e., a light-induced, cytochrome b -559-dependent, cyclic electron transport pathway around PSII that promotes translocation of protons from plastoquinol into the aqueous domain (lumen) of photosynthetic membranes (thylakoids). Light-induced proton transport coupled to light-induced electron transport is an essential aspect of energy transduction in photosynthesis because it generates an electrochemical proton gradient that drives ATP synthesis by the process of photosynthetic phosphorylation. The principal carrier of electrons and protons in thylakoids is the plastoquinone/plastoquinol couple. We propose that the b -559 cycle functions as a redox-linked proton pump that may operate jointly with the Rieske iron-sulfur pathway in oxidizing plastoquinol. The overall effect of such concerted oxidation of plastoquinol would be the translocation into the thylakoid lumen of two protons for each electron transferred from water to plastocyanin via plastoquinone.

The photosynthetic apparatus converts light into chemical energy by a series of reactions that give rise to a coupled flow of electrons and protons that generate reducing power and ATP, respectively. A key intermediate in these reactions is plastoquinone (PQ), the most abundant electron and proton (hydrogen) carrier in photosynthetic membranes (thylakoids). PQ ultimately transfers electrons to a terminal electron acceptor by way of the Rieske Fe-S center of the cytochrome bf complex. In the absence of a terminal acceptor, electrons accumulate in the PQ pool, which is reduced to plastoquinol (PQH(2)), and also on a specialized PQ, Q(A), which is reduced to an unprotonated semiquinone anion (Q(A) (-)). The accumulation of Q(A) (-) is measured by a rise in fluorescence yield and the accumulation of PQH(2) is measured by absorption difference spectrometry. We have found that in the absence of a terminal electron acceptor, two chemically diverse proton-conducting ionophores (protonophores), 2,6-di-t-butyl-4-(2',2'-dicyanovinyl)phenol (SF 6847) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), induced oxidation of PQH(2) and quenching of chloroplast fluorescence, signifying oxidation of Q(A) (-). The two protonophores produced the same effects even when the only recognized pathway of PQH(2) oxidation by way of the cytochrome bf complex was inhibited by dibromothymoquinone. Two other uncouplers, gramicidin and nigericin, which are not protonophores but facilitate proton movement across membranes by other mechanisms, were ineffective. These findings are consistent with the operation in the oxygen-generating photosystem (photosystem II) of a cyclic, proton-conducting pathway.

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/ b 6 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.

The discovery of photosynthetic phosphorylation (photophosphorylation) revealed the capacity of the photosynthetic apparatus to convert the electromagnetic energy of sunlight into the biochemical energy of ATP. Two types were found: cyclic photophosphorylation in which ATP is the sole product of energy conversion and noncyclic photophosphorylation in which ATP formation is accompanied by oxygen evolution and the generation of reducing power whose carrier is ferredoxin.

New evidence is presented in support of the concept that reducing power for photosynthesis is generated solely by photosystem II (the oxygenic photosystem) when it transfers electrons from water to ferredoxin without the collaboration of photosystem I, the anoxygenic photosystem responsible for cyclic photophosphorylation. Membrane vesicles of opposite sidedness were prepared from spinach chloroplasts by the two-phase partition method: inside-out-vesicles greatly enriched in photosystem II and right-side-out vesicles containing both photosystems and having the same sidedness orientation as unfractionated chloroplast membranes. In both types of vesicles, plastoquinone analogues were used to inhibit light-induced electron transport from water to ferredoxin and from water to native photosystem I acceptors, the membrane-bound iron-sulfur centers A and B. In right-side-out vesicles the photoreduction of iron-sulfur centers A and B was more sensitive to plastoquinone inhibitors than the photoreduction of ferredoxin, whereas the converse was found in inside-out vesicles in which a greatly enhanced sensitivity of ferredoxin reduction to plastoquinone inhibitors was detected: the photoreduction of ferredoxin was about 80% inhibited at low concentrations of plastoquinone inhibitors that had practically no effect on the photoreduction of iron-sulfur centers A and B. These findings appear to exclude the possibility that these photosystem I contaminants were involved in the photoreduction of ferredoxin by the PSII-enriched inside-out vesicles.

It is now widely held that the light-induced noncyclic (linear) electron transport from water to NADP(+) requires the collaboration in series of the two photosystems that operate in oxygen-evolving cells: photosystem II (PSII) photooxidizes water and transfers electrons to photosystem I (PSI); PSI photoreduces ferredoxin, which in turn reduces NADP(+) (the Z scheme). However, a recently described alternative scheme envisions that PSII drives the noncyclic electron transport from water to ferredoxin and NADP(+) without the collaboration of PSI, whose role is limited to cyclic electron transport [Arnon, D. I., Tsujimoto, H. Y. & Tang, G. M.-S. (1981) Proc. Natl. Acad. Sci. USA 78, 2942-2946]. Reported here are findings at variance with the Z scheme and consistent with the alternative scheme. Thylakoid membrane vesicles were isolated from spinach chloroplasts by the two-phase aqueous polymer partition method. Vesicles, originating mainly from appressed chloroplast membranes that are greatly enriched in PSII, were turned inside-out with respect to the original sidedness of the membrane. With added plastocyanin, ferredoxin, and ferredoxin-NADP(+) reductase, the inside-out vesicles enriched in PSII gave a significant photoreduction of NADP(+) with water as electron donor, under experimental conditions that appear to exclude the participation of PSI.

The SciencesVolume 22, Issue 7 p. 22-27 Sunlight, Earth Life Daniel I. Arnon, Daniel I. Arnon Daniel I. Arnon, professor of cell physiology, and research biochemist at the University of California, Berkeley, received the National Medal of Science, in 1973, for “fundamental research into the mechanism by which green plants utilize light to produce chemical energy and oxygen.”Search for more papers by this author Daniel I. Arnon, Daniel I. Arnon Daniel I. Arnon, professor of cell physiology, and research biochemist at the University of California, Berkeley, received the National Medal of Science, in 1973, for “fundamental research into the mechanism by which green plants utilize light to produce chemical energy and oxygen.”Search for more papers by this author First published: October 1982 https://doi.org/10.1002/j.2326-1951.1982.tb02101.xCitations: 20 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume22, Issue7October 1982Pages 22-27 RelatedInformation

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 78, 2942–2946, 1981).

The currently prevalent concept of the generation of photosynthetic reducing power in oxygen-evolving cells envisions a linear (noncyclic) electron flow from water to ferredoxin (and thence to NADP + ) that requires the collaboration of photosystems I and II (PSI and PSII) joined by plastoquinone and other electron carriers (the Z scheme). The essence of the Z scheme is that only PSI can reduce ferredoxin—i.e., that, after being energized to an intermediate reducing potential by PSII, electrons from water are transported via plastoquinone to PSI which energizes the electrons to their ultimate reducing potential adequate for the reduction of ferredoxin. Basic to the Z scheme is the function of plastoquinone as the obligatory link in electron transport from PSII to PSI. However, we have found that, when plastoquinone function was inhibited, ferredoxin was photoreduced by water without the collaboration of PSI. We now report evidence for an important function of plastoquinone in the translocation of protons liberated inside the thylakoid membrane by photooxidation of water. When the oxygenic photoreduction (i.e., by water) of ferredoxin was blocked by plastoquinone inhibitors, dibromothymoquinone or dinitrophenol ether of iodonitrothymol, the photoreduction of ferredoxin was restored by each of four chemically diverse uncouplers, similar only in their ability to facilitate proton movement across membranes. Similar results were obtained for the oxygenic reduction of NADP + . Our results suggest that the light-induced electron flow from water cannot be maintained unless the simultaneously liberated protons are removed from inside the membrane via plastoquinone. The new evidence is embodied in a concept of an oxygenic photosystem for photosynthetic electron and proton transport, which we propose as an alternative to the Z scheme, to account for photoreduction of ferredoxin-NADP + by water and the coupled oxygenic (formerly noncyclic) ATP formation without involving PSI. The role of the anoxygenic photosystem (formerly called PSI) is ATP formation by cyclic photophosphorylation.

Pairs of two molecular species of soluble chloroplast-type ferredoxins (Fd I and Fd II) from Nostoc muscorum and Aphanothece sacrum were used to examine and compare the abilities of ferredoxin to substitute for spinach ferredoxin in the photoreduction of NADP+ by spinach chloroplasts or N. muscorum membrane fragments and to link the reducing power of illuminated spinach chloroplasts to the Bacillus polymyxa nitrogenase system. Ferredoxins II of Nostoc and Aphanothece showed rather low activities in NADP+ photoreduction and nitrogenase system with spinach chloroplasts as the photosensitizer, compared to other ferredoxins. However, there was no difference between two ferredoxins (Fd I and Fd II) from Nostoc in NADP+ photoreduction by photosynthetic membrane fragments prepared from the same organism, N. muscorum. The biological significance of two molecular species of ferredoxins in one organism could be ascribed to the different contribution of each ferredoxin to certain biological reactions in which ferredoxin functioned as an electron carrier.

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.

FEBS LettersVolume 120, Issue 1 p. 119-124 Full-length articleFree Access Photoreduction of ferredoxin by chloroplasts with or without an accompanying photoreduction of the bound iron—sulfur centers Contrasting effects of electron donors to photosystems I and II Daniel I. Arnon, Daniel I. Arnon Department of Cell Physiology, University of California, Berkeley, CA 94720, USASearch for more papers by this authorHarry Y. Tsujimoto, Harry Y. Tsujimoto Department of Cell Physiology, University of California, Berkeley, CA 94720, USASearch for more papers by this authorGeorge M.-S. Tang, George M.-S. Tang Department of Cell Physiology, University of California, Berkeley, CA 94720, USASearch for more papers by this author Daniel I. Arnon, Daniel I. Arnon Department of Cell Physiology, University of California, Berkeley, CA 94720, USASearch for more papers by this authorHarry Y. Tsujimoto, Harry Y. Tsujimoto Department of Cell Physiology, University of California, Berkeley, CA 94720, USASearch for more papers by this authorGeorge M.-S. Tang, George M.-S. Tang Department of Cell Physiology, University of California, Berkeley, CA 94720, USASearch for more papers by this author First published: October 20, 1980 https://doi.org/10.1016/0014-5793(80)81060-XCitations: 7AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume120, Issue1October 20, 1980Pages 119-124 ReferencesRelatedInformation

An investigation by paramagnetic resonance spectroscopy of the photoreduction of ferredoxin, oxygenically by water and anoxygenically by a direct electron donor to photosystem I, led to the unexpected findings that different reductive mechanisms may be involved. Ferredoxin photoreduced by water was not reoxidized in the light under aerobic conditions and, under anaerobic conditions, it was remarkably resistant to reoxidation in the dark. By contrast, ferredoxin photoreduced by a donor to photosystem I was readily reoxidized in the light by air and, under anaerobic conditions, by exposure to darkness. Furthermore, when electron transport linking photosystems I and II was inhibited by a plastoquinone antagonist, ferredoxin was photoreduced by water with no evidence for an accompanying photoreduction of the more electronegative bound iron-sulfur centers in chloroplasts. These findings are at variance with the now prevalent concepts of photosynthetic electron transport.

Acetylene reduction by nitrogenase from Rhodospirillum rubrum , unlike that by other nitrogenases, was recently found by other investigators to require an activation of the iron protein of nitrogenase by an activating system comprising a chromatophore membrane component, adenosine 5′-triphosphate (ATP), and divalent metal ions. In an extension of this work, we observed that the same activating system was also required for nitrogenase-linked H 2 evolution. However, we found that, depending on their nitrogen nutrition regime, R. rubrum cells produced two forms of nitrogenase that differed in their Fe protein components. Cells whose nitrogen supply was totally exhausted before harvest yielded predominantly a form of nitrogenase (A) whose enzymatic activity was not governed by the activating system, whereas cells supplied up to harvest time with N 2 or glutamate yielded predominantly a form of nitrogenase (R) whose enzymatic activity was regulated by the activating system. An unexpected finding was the rapid (less than 10 min in some cases) intracellular conversion of nitrogenase A to nitrogenase R brought about by the addition to nitrogen-starved cells of glutamine, asparagine, or, particularly, ammonia. This finding suggests that mechanisms other than de novo protein synthesis were involved in the conversion of nitrogenase A to the R form. The molecular weights of the Fe protein and Mo-Fe protein components from nitrogenases A and R were the same. However, nitrogenase A appeared to be larger in size, because it had more Fe protein units per Mo-Fe protein than did nitrogenase R. A distinguishing property of the Fe protein from nitrogenase R was its ATP requirement. When combined with the Mo-Fe protein (from either nitrogenase A or nitrogenase R), the R form of Fe protein required a lower ATP concentration but bound or utilized more ATP molecules during acetylene reduction than did the A form of Fe protein. No differences between the Fe proteins from the two forms of nitrogenase were found in the electron paramagnetic resonance spectrum, midpoint oxidation-reduction potential, or sensitivity to iron chelators.

FEBS LettersVolume 98, Issue 2 p. 381-385 Full-length articleFree Access Photoreduction of membrane-bound paramagnetic component X by water as electron donor Tetsuo Hiyama, Tetsuo Hiyama Department of Cell Physiology, University of California, Berkeley, CA 94720, USASearch for more papers by this authorHarry Y. Tsujimoto, Harry Y. Tsujimoto Department of Cell Physiology, University of California, Berkeley, CA 94720, USASearch for more papers by this authorDaniel I. Arnon, Daniel I. Arnon Department of Cell Physiology, University of California, Berkeley, CA 94720, USASearch for more papers by this author Tetsuo Hiyama, Tetsuo Hiyama Department of Cell Physiology, University of California, Berkeley, CA 94720, USASearch for more papers by this authorHarry Y. Tsujimoto, Harry Y. Tsujimoto Department of Cell Physiology, University of California, Berkeley, CA 94720, USASearch for more papers by this authorDaniel I. Arnon, Daniel I. Arnon Department of Cell Physiology, University of California, Berkeley, CA 94720, USASearch for more papers by this author First published: February 15, 1979 https://doi.org/10.1016/0014-5793(79)80222-7Citations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 R. Malkin, A.J. Bearden, Proc. Natl. Acad. Sci. USA, 68, (1971), 16– 19. 2 M.C.W. Evans, A. Telfer, A.V. Lord, Biochim. Biophys. Acta, 267, (1972), 530– 537. 3 M.C.W. Evans, A. Telfer, A.V. Lord, Biochem. Biophys. Res. Commun., 51, (1973), 593– 596. 4 M.C.W. Evans, S.G. Reeves, R. Cammack, FEBS Lett., 49, (1974), 111– 114. 5 A.R. McIntosh, M. Chu, J.R. Bolton, Biochim. Biophys. Acta, 376, (1975), 308– 314. 6 A.R. McIntosh, J.R. Bolton, Biochim. Biophys. Acta, 430, (1976), 555– 559. 7 M.C.W. Evans, C.K. Sihra, J.R. Bolton, R. Cammack, Nature, 256, (1975), 668– 670. 8 M.C.W. Evans, C.K. Sihra, R. Cammack, Biochem. J., 158, (1976), 71– 77. 9 P. Heathcote, D.L. Williams-Smith, M.C.W. Evans, Biochem. J., 170, (1978), 373– 378. 10 P. Heathcote, D.L. Williams-Smith, C.K. Sihra, M.C.W. Evans, Biochim. Biophys. Acta, 503, (1978), 333– 342. 11 K. Sauer, P. Mathis, S. Acker, J.A. Van Best, Biochim. Biophys. Acta, 503, (1978), 120– 134. 12 V.A. Shuvalov, E. Dolan, B. Ke, Proc. Natl. Acad. Sci. USA, (1979), in press 13 B. Ke, E. Dolan, K. Sugahara, F.M. Hawkridge, S. Demeter, E.R. Shaw, Photosynthetic Organelles. S. Miyachi Plant Cell Physiol. 3, (1977), 187– 199. spec. iss. 14 B. Ke, R.E. Hansen, H. Beinert, Proc. Natl. Acad. Sci. USA, 70, (1973), 2941– 2945. 15 D.I. Arnon, H.Y. Tsujimoto, T. Hiyama, Proc. Natl. Acad. Sci. USA, 74, (1977), 3826– 3830. 16 D.I. Arnon, R.K. Chain, Proc. Natl. Acad. Sci. USA, 72, (1975), 4956– 4961. 17 D.I. Arnon, B.D. McSwain, H.Y. Tsujimoto, K. Wada, Biochim. Biophys. Acta, 357, (1974), 231– 245. errata D.I. Arnon, B.D. McSwain, H.Y. Tsujimoto, K. Wada, Biochim. Biophys. Acta, 368, (1974), 459– 18 D.I. Arnon, Plant Physiol., 24, (1949), 1– 15. 19 M. Losada, D.I. Arnon, H.W. Linskens Modern Methods of Plant Analysis 7, (1964), Springer-Verlag Berlin 569– 615. 20 M. Shin, K. Tagawa, D.I. Arnon, Biochem. Z., 338, (1963), 84– 96. 21 E.H. Evans, R. Cammack, M.C.W. Evans, Biochem. Biophys. Res. Commun., 68, (1976), 1212– 1218. 22 D.I. Arnon, R.K. Chain, Photosynthetic Organelles. S. Miyachi Plant Cell Physiol. 3, (1977), 129– 147. spec. iss. 23 R.K. Chain, D.I. Arnon, Proc. Natl. Acad. Sci. USA, 74, (1977), 3377– 3381. 24 D.I. Arnon, R.K. Chain, FEBS Lett., 82, (1977), 297– 302. 25 T. Hiyama, B. Ke, Proc. Natl. Acad. Sci. USA, 68, (1971), 1010– 1013. 26 T. Hiyama, B. Ke, G. Forti Proc. 2nd Int. Cong. Photosynthesis Res. (1972), Dr W. Junk NV., The Hague 491– 497. 27 T. Hiyama, B. Ke, Arch. Biochem. Biophys., 147, (1971), 99– 108. Citing Literature Volume98, Issue2February 15, 1979Pages 381-385 ReferencesRelatedInformation