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The inhibitor methyl viologen (MV) has been widely used in photosynthesis to study oxidative stress. Its effects on electron transfer kinetics in Synechocystis sp. PCC6803 cells were studied to characterize its electron-accepting properties. For the first hundreds of flashes following MV addition at submillimolar concentrations, the kinetics of NADPH formation were hardly modified (less than 15% decrease in signal amplitude) with a significant signal decrease only observed after more flashes or continuous illumination. The dependence of the P700 photooxidation kinetics on the MV concentration exhibited a saturation effect at 0.3 mM MV, a concentration which inhibits the recombination reactions in photosystem I. The kinetics of NADPH formation and decay under continuous light with MV at 0.3 mM showed that MV induces the oxidation of the NADP pool in darkness and that the yield of linear electron transfer decreased by only 50% after 1.5–2 photosystem-I turnovers. The unexpectedly poor efficiency of MV in inhibiting NADPH formation was corroborated by in vitro flash-induced absorption experiments with purified photosystem-I, ferredoxin and ferredoxin-NADP + -oxidoreductase. These experiments showed that the second-order rate constants of MV reduction are 20 to 40-fold smaller than the competing rate constants involved in reduction of ferredoxin and ferredoxin-NADP + -oxidoreductase. The present study shows that MV, which accepts electrons in vivo both at the level of photosystem-I and ferredoxin, can be used at submillimolar concentrations to inhibit recombination reactions in photosystem-I with only a moderate decrease in the efficiency of fast reactions involved in linear electron transfer and possibly cyclic electron transfer. [ABSTRACT FROM AUTHOR]

Photosynthetic [2Fe-2S] plant-type ferredoxins have a central role in electron transfer between the photosynthetic chain and various metabolic pathways. Several genes are coding for [2Fe 2S] ferredoxins in cyanobacteria, with four in the thermophilic cyanobacterium Thermosynechococcus elongatus. The structure and functional properties of the major ferredoxin Fd1 are well known but data on the other ferredoxins are scarce. We report the structural and functional properties of a novel minor type ferredoxin, Fd2 of T. elongatus , homologous to Fed4 from Synechocystis sp. PCC 6803. Remarkably, the midpoint potential of Fd2, Em = −440 mV, is lower than that of Fd1, Em = −372 mV. However, while Fd2 can efficiently react with photosystem I or nitrite reductase, time-resolved spectroscopy shows that Fd2 has a very low capacity to reduce ferredoxin-NADP+ oxidoreductase (FNR). These unique Fd2 properties are discussed in relation with its structure, solved at 1.38 Å resolution. The Fd2 structure significantly differs from other known ferredoxins structures in loop 2, N-terminal region, hydrogen bonding networks and surface charge distributions. UV–Vis, EPR, and Mid- and Far-IR data also show that the electronic properties of the [2Fe 2S] cluster of Fd2 and its interaction with the protein differ from those of Fd1 both in the oxidized and reduced states. The structural analysis allows to propose that valine in the motif Cys 53 ValAsnCys 56 of Fd2 and the specific orientation of Phe72, explain the electron transfer properties of Fd2. Strikingly, the nature of these residues correlates with different phylogenetic groups of cyanobacterial Fds. With its low redox potential and its discrimination against FNR, Fd2 exhibits a unique capacity to direct efficiently photosynthetic electrons to metabolic pathways not dependent on FNR. • The first structure of an alternative [2Fe 2S] ferredoxin, Fd2, was resolved. • Fd2 significantly differs from the main photosynthetic ferredoxins. • Fd2 efficiently reacts with photosystem I and nitrite reductase. • Despite its low midpoint potential (−440 mV), Fd2 is unable to reduce FNR. • The specific role of amino acids in Fd2 properties and Fd phylogeny is discussed. [ABSTRACT FROM AUTHOR]

Photosynthetic ferredoxin and its main partner ferredoxin–NADP + -reductase (FNR) are key proteins during the photoproduction of reductive power involved in photosynthetic growth. In this work, we used covalent attachment of ruthenium derivatives to different cysteine mutants of ferredoxin to trigger by laser-flash excitation both ferredoxin reduction and subsequent electron transfer from reduced ferredoxin to FNR. Rates and yields of reduction of the ferredoxin [2Fe–2S] cluster by reductively quenched Ru* could be measured for the first time for such a low redox potential protein whereas ferredoxin–FNR electron transfer was characterized in detail for one particular Ru–ferredoxin covalent adduct. For this adduct, the efficiency of FNR single reduction by reduced ferredoxin was close to 100% under both first-order and diffusion-limited second-order conditions. Interprotein intracomplex electron transfer was measured unambiguously for the first time with a fast rate of c. 6500 s − 1 . Our measurements point out that Ru photosensitizing is a powerful approach to study the functional interactions of ferredoxin with its numerous partners besides FNR. [ABSTRACT FROM AUTHOR]