George M.-S. Tang

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

This communication reports new evidence consonant with a recent finding [l] of ferredoxin photoreduction by water when the photoreduction of the bound iron-sulfur centers associated with photosystem I of chloroplasts [2-41 was strongly inhibited. Chloroplasts photoreduce ferredoxin by electrons that originate from water, via photosystem II, or by electrons supplied directly to photosystem I by artificial donors that bypass photosystem II [S]. Contrary to expectations, only with a direct donor to photosystem I (ascorbate/DCIP) was the photoreduction of ferredoxin by chloroplasts [l], like the photoreduction of ferredoxin by cyanobacterial membrane fragments [6], unequivocally associated with the photoreduction of the bound iron-sulfur centers. With water as donor, ferredoxin was found in [ 1 ] to be in a predominantly reduced steady state, even when the photoreduction of the bound iron-sulfur centers was inhibited by DBMIB (2,5-dibromo-3methyl&isopropyl-l ,Cbenzoquinone). DBMIB is an antagonist of plastoquinone, the chloroplast component deemed essential for electron transfer from photosystem II to photosystem I [7,8]. It appeared, therefore, that, with water as the electron donor, the photoreduction of the bound iron-sulfur centers by photosystem I was an accompaniment but not a prerequisite for the photoreduction of ferredoxin. Normally, electrons originating

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 78, 2942–2946, 1981).

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

Abstract 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.

Photoreduction of NADP+ by a chloroplast photosystem II preparation: effect of light intensity

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.

Inhibition by plastoquinone analogues of ferricyanide reduction by a photosystem II chloroplast preparation

Abstract There is wide agreement that inhibition of plastoquinone function by plastoquinone analogues, of which the best known is dibromothymoquinone (DBMIB), inhibits electron transport from Photosystem II (PS II) to Photosystem I (PS I), but does not inhibit electron transport that is dependent on PS II alone. In contrast, we have recently presented evidence in support of a hypothesis for a hitherto unrecognized additional role of plastoquinone in the conductance of protons derived from the photooxidation of water by PS II (Arnon, D.I. and Tang, G.M.-S. (1985) Biochim. Biophys. Acta 809, 167–172). Since the transport of water-derived electrons and protons is coupled, this hypothesis predicts that photosynthetic electron transport by PS II alone would still be sensitive to DBMIB inhibition. We now report that this prediction has been verified by obtaining inhibition by low concentrations of DBMIB of electron transport from water to ferricyanide in a PS II preparation depleted of plastocyanin and PS I. The preparation consisted of inside-out vesicles isolated by the aqueous polymer two-phase partition method. The pattern of DBMIB inhibition in the PS II preparation was the same as that in unfractionated chloroplasts: inhibition of ferricyanide reduction was relieved by the addition of catalytic amounts of one of several lipophilic acceptors that appear to bypass the site of DBMIB inhibition at the Rieske FeS center of the cytochrome f b 6 complex. The bearing of these findings on the role of plastoquinones in the conductance of protons released by the photooxidation of water is discussed.

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

Abstract 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.

Enhanced sensitivity to plastoquinone inhibitors of ferredoxin photoreduction by photosystem II in inside-out chloroplast vesicles

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.