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 PSU that promotes translocation of protons from plastoquinol into the aqueous domain (lumen) of photosynthetic membranes (thylakoids). Light-induced proton transport coupled to lightinduced 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.Light-induced proton transport, coupled to light-induced electron transport, gained prominence in photosynthesis research with the recognition that it produces in photosynthetic membranes (thylakoids) an electrochemical proton gradient (A/.tH+) that drives ATP synthesis (1) in the process of photosynthetic phosphorylation (2). This process consists of cyclic (anoxygenic) photophosphorylation in which ATP is the sole product and noncyclic (oxygenic) photophosphorylation in which ATP formation is accompanied by oxygen evolution and the generation ofreducing power whose carrier is ferredoxin (2, 3). Oxygen apart, ATP and reduced ferrodoxin are the two products of transduction of sunlight's electromagnetic energy into forms of chemical energy that, directly or through intermediates, drive the biosynthetic and regulatory reactions of photosynthesis including, but not limited to, CO2 assimilation (4).In noncyclic, oxygenic photophosphorylation, A/.H+ is generated by protons released from two sources: the photooxidation of water (see Discussion) and the oxidation of plastoquinol (PQH2). As for PQH2, the accepted view is that thylakoids can oxidize it only via the Rieske iron-sulfur center pathway that is sensitive to inhibition by dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) (5, 6). However, evidence was recently obtained (7) that PQH2 is also photooxidized by an alternative, cyclic pathway not inhibited by DBMIB. The cyclic pathway was uncovered through the use of uncouplers, specifically proton conductors (protonophores). Two chemically diverse photonophores, 2,6-di-(t-butyl)-4-(2',2'-dicyanovinyl)phenol (SF 6847) (8,9) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) (10, 11), induced oxidation of PQH2 and dramatically lowered...

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 11 (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 PSIl 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 offerredoxin-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.Photooxidation of water, 2H20-) 4 e-+ 4H + 02, is a key reaction in plant photosynthesis. Aside from its all-important by-product, oxygen, the liberated electrons eventually reduce ferredoxin (1, 2) and then NADP+ (3), thereby accounting for photosynthetic reducing power; the liberated protons contribute to the protonmotive force (4, 5) which accounts for the ("noncyclic") ATP formation that is coupled to NADP+ reduction (6).Photooxidation of water takes place inside the thylakoid membranes (7). These contain two photocenters identified with photosystems I and II (PSI and PSII) and a chain of carriers responsible for the transport ofelectrons and protons within and across the membrane. According to the now-prevalent concepts embodied in the so-called Z scheme (8, 9), PSII photooxidizes water but cannot reduce ferredoxin because it can energize the released electrons only to an intermediate potential; the electrons are transported from PSII t...

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 bfcomplex. In the absence of a terminal acceptor, electrons accumulate in the PQ pool, which is reduced to plastoquinol (PQH2), and also on a specialized PQ, QA, which is reduced to an unprotonated semiquinone anion (Q-). The accumulation of Q-is measured by a rise in fluorescence yield and the accumulation of PQH2 is measured by absorption difference spectrometry. We have found that in the absence of a terminal electron acceptor, two chemically diverse protonconducting ionophores (protonophores), 2,6-di-t-butyl-4-(2',2'-dicyanovinyl)phenol (SF 6847) and carbonylcyanide ptrifluoromethoxyphenylhydrazone (FCCP), induced oxidation of PQH2 and quenching of chloroplast fluorescence, signifying oxidation of Q-. The two protonophores produced the same effects even when the only recognized pathway of PQH2 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 recently described perspective on photosynthesis envisions the operation in the oxygen-generating photosystem (photosystem II; PSII) of a light-induced cyclic pathway for conductance of protons (1). We now report effects of uncouplers on chloroplast fluorescence and the redox state of plastoquinone (PQ) that are consistent with the operation of such a pathway.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 (2). A key intermediate in these reactions is PQ, the most abundant redox component in photosynthetic membranes (thylakoids) (3, 4). Because PQ-plastoquinol (PQH2) oxidoreductions (PQ + 2 e-+ 2 H+ ;± PQH2) involve transfers of hydrogen atoms (electrons plus protons), PQ is both the main electron and the main proton carrier in thylakoids.In functioning chloroplasts, PQH2 is ultimately oxidized by a terminal electron acceptor, by way of the Rieske Fe-S center of the cytochrome bfcomplex (5). In the absence of a terminal acceptor, PQH2 accumulates; electrons then "back up" and accumulate on a specialized PQ (QA). The accumulation of QA is monitored by a rise in variable fluorescence (Fv) (6).We have found that in the absence of a terminal acceptor, two chemically diverse proton-conducting ionophores (uncouplers...

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 PSI1 drives the noncyclic electron transport from water to ferredoxin and NADP+ without the collaboration of PSI, whose role is lim- 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.It is now well established (1) that the photosynthetic apparatus of plants utilizes two photosystems, photosystem I (PSI) and photosystem II (PSII), for the conversion of the electromagnetic energy of sunlight into biologically useful chemical energy via two processes: cyclic photophosphorylation that generates only ATP (2, 3) and noncyclic photophosphorylation that concurrently generates ATP and reducing power, the carrier of which was identified as NADPH (4) and later as reduced ferredoxin (5, 6). Reduced ferredoxin is the versatile carrier of photosynthetically generated energy-rich electrons destined for many targets (7), including the enzymatic reduction of NADP+ (8, 9) and the regulation of cyclic photophosphorylation (10).The source of electrons needed for the light-induced reduction of ferredoxin is the photooxidation of water, a reaction that also produces molecular oxygen and contributes protons used to generate an electrochemical proton gradient (11, 12) responsible for the (noncyclic) ATP formation coupled to ferredoxin (6) or NADP+ (4) reduction. There is wide agreement on two points: (i) photooxidation of water is driven by PSII (1) and (ii) cyclic photosphosphorylation is driven by PSI (1, 13).There is also agreement that PSI is capable of reducing ferredoxin and NADP+ by artificial reductants (14), but there is disagreement as to whether PSI is responsible for the reduction of ferredoxin under physiological conditions-i. e., bv electrons liberated in the photooxidation of water by PSI.Most investigators favor the so-called Z scheme, according to which only PSI is competent to photoreduce ferredoxin, and a collaboration of PSII and PSI is therefore required for the complete noncyclic electron transport from water to ferredoxin (1, 14). However, a recently described alternative concept (15...

Recent work in this and other laboratories has demonstrated that, contrary to the favored Z scheme hypothesis, photosystern 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.