Protonophores induce plastoquinol oxidation and quench chloroplast fluorescence: Evidence for a cyclic, proton-conducting pathway in oxygenic photosynthesis

Abstract

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...

Modelling the fast fluorescence rise of photosynthesis

“…Perhaps PS I is not the most likely candidate, at least not in its classical role in linear electron transport. This is corroborated by the observation that the typical ID dip also occurs in the presence of DBMIB (McCauley et al, 1987), an inhibitor which blocks the reoxidation of PQ.…”

“…The possibility of cyclic electron transport around PS II proposed by Arnon and Tang (1988) and McCauley et al (1987) remains a great challenge to the common picture of linear electron transport.…”

Fluorescence as a Tool to Understand Changes in Photosynthetic Electron Flow Regulation

“…Typical concentrations of FCCP are 1-10 mM (Shyam et al 1993;Sigalat et al 1993;Singh et al 1996). At low concentrations FCCP quenches PSII fluorescence, indicative of the reoxidation of Q A − (McCauley et al 1987), while it requires much higher concentrations to perform in its function as an uncoupler of oxidative phosphorylation (Canaani and Havaux 1990). When incubated with cells under photoinhibitory light, FCCP accelerates photoinhibition and rapidly quenches fluorescence yield (McCauley et al 1987;Shyam et al 1993;Singh et al 1996).…”

“…Uncouplers function by dissociating electron transport from ATP synthesis during photosynthetic phosphorylation (Moreland 1980;McCauley et al 1987). This is accomplished by dissipating the energised state (H + ) of the membrane (DpH) before the energy can be utilised in ADP phosphorylation (Moreland 1980) and thus prevent the formation of the trans-thylakoid DpH gradient.…”

Photoreduction of silicomolybdate in chloroplasts by agents accelerating the deactivation reactions of the water‐oxidizing system

“…On the other hand, uncouplers FCCP and 2,6-di(t-butyl)-4-(2',2'-dicyanovinyl)phenol (SF 6847) induced the oxidation of plastoquinol and the oxidation of cytochrome b-559 that appeared to be part of the cyclic pathway for the oxidation of plastoquinol in the PSII [17,18]. As was noted by Arnon and Tang [18], the uncouplers "must have acted not as electron acceptors but as promoters of the oxidation of cytochrome b-559 by virtue of their special capacity for reversible binding of protons".…”