Photoreduction and Reoxidation of the Three Iron-Sulfur Clusters of Reaction Centers of Green Sulfur Bacteria

AbstractIron-sulfur clusters are the terminal electron acceptors of the photosynthetic reaction centers of green sulfur bacteria and photosystem I. We have studied electron-transfer reactions involving these clusters in the green sulfur bacterium Chlorobium tepidum, using flash-absorption spectroscopic measurements. We show for the first time that three different clusters, named FX, F1, and F2, can be photoreduced at room temperature during a series of consecutive flashes. The rates of electron escape to exogenous acceptors depend strongly upon the number of reduced clusters. When two or three clusters are reduced, the escape is biphasic, with the fastest phase being 12–14-fold faster than the slowest phase, which is similar to that observed after single reduction. This is explained by assuming that escape involves mostly the second reducible cluster. Evidence is thus provided for a functional asymmetry between the two terminal acceptors F1 and F2. From multiple-flash experiments, it was possible to derive the intrinsic recombination rates between P840+ and reduced iron-sulfur clusters: values of 7, 14, and 59s−1 were found after one, two and three electron reduction of the clusters, respectively. The implications of our results for the relative redox potentials of the three clusters are discussed

Pre-steady-state kinetic studies of redox reactions catalysed by Bacillus subtilis ferredoxin-NADP+ oxidoreductase with NADP+/NADPH and ferredoxin

Ferredoxin-NADP+ oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP+, NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP+ and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNRox)-NADPH and reduced FNR (FNRred)-NADP+, both having CT absorption bands centered at approximately 600 nm. After mixing BsFNRox with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNRred was mixed with NADP+, the amount of BsFNRox increased with increasing NADP+ concentration, but BsFNRred remained as the major species at equilibrium even with about 50-fold molar excess NADP+. In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~ 500 s- 1) was much higher than the reverse one (< 10 s- 1). Mixing BsFdred with BsFNRox induced rapid formation of a neutral semiquinone form. This process was almost completed within 1 ms. Subsequently the neutral semiquinone form was reduced to the hydroquinone form with an apparent rate constant of 50 to 70 s- 1 at 10 °C, which increased as BsFdred increased from 40 to 120 μM. The reduction rate of BsFNRox by BsFdred was markedly decreased by premixing BsFNRox with BsFdox, indicating that the dissociation of BsFdox from BsFNRsq is rate-limiting in the reaction. The characteristics of the BsFNR reactions with NADP+/NADPH were compared with those of other types of FNRs. © 2016 Elsevier B.V. All rights reserved.Embargo Period 12 month

Breeding for increased nitrogen-use efficiency: a review for wheat (T. aestivum L.)

Nitrogen fertilizer is the most used nutrient source in modern agriculture and represents significant environmental and production costs. In the meantime, the demand for grain increases and production per area has to increase as new cultivated areas are scarce. In this context, breeding for an efficient use of nitrogen became a major objective. In wheat, nitrogen is required to maintain a photosynthetically active canopy ensuring grain yield and to produce grain storage proteins that are generally needed to maintain a high end-use quality. This review presents current knowledge of physiological, metabolic and genetic factors influencing nitrogen uptake and utilization in the context of different nitrogen management systems. This includes the role of root system and its interactions with microorganisms, nitrate assimilation and its relationship with photosynthesis as postanthesis remobilization and nitrogen partitioning. Regarding nitrogen-use efficiency complexity, several physiological avenues for increasing it were discussed and their phenotyping methods were reviewed. Phenotypic and molecular breeding strategies were also reviewed and discussed regarding nitrogen regimes and genetic diversity

Electron-transfer kinetics in cyanobacterial cells: methyl viologen is a poor inhibitor of linear electron flow.

International audienceThe 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

Dynamics and energetics of cyanobacterial photosystem I:ferredoxin complexes in different redox states

Fast turnover of ferredoxin/Fd reduction by photosystem-I/PSI requires that it dissociates rapidly after it has been reduced by PSI:Fd intracomplex electron transfer. The rate constants of Fd dissociation from PSI have been determined by flash-absorption spectroscopy with different combinations of cyanobacterial PSIs and Fds, and different redox states of Fd and of the terminal PSI acceptor (FAFB). Newly obtained values were derived firstly from the fact that the dissociation constant between PSI and redox-inactive gallium-substituted Fd increases upon (FAFB) reduction and secondly from the characterization and elucidation of a kinetic phase following intracomplex Fd reduction to binding of oxidized Fd to PSI, a process which is rate-limited by the foregoing dissociation of reduced Fd from PSI. By reference to the complex with oxidized partners, dissociation rate constants were found to increase moderately with (FAFB) single reduction and by about one order of magnitude after electron transfer from (FAFB)(-) to Fd, therefore favoring turnover of Fd reduction by PSI. With Thermosynechococcus elongatus partners, values of 270, 730 and \textgreater10000 s(-1) were thus determined for (FAFB)Fdoxidized, (FAFB)(-)Fdoxidized and (FAFB)Fdreduced, respectively. Moreover, assuming a conservative upper limit for the association rate constant between reduced Fd and PSI, a significant negative shift of the Fd midpoint potential upon binding to PSI has been calculated (\textless-60 mV for Thermosynechococcus elongatus). From the present state of knowledge, the question is still open whether this redox shift is compatible with a large (\textgreater10) equilibrium constant for intracomplex reduction of Fd from (FAFB)(-)

Ferredoxin Reduction by Photosystem I fromSynechocystissp. PCC 6803: Toward an Understanding of the Respective Roles of Subunits PsaD and PsaE in Ferredoxin Binding

The process of ferredoxin reduction by photosystem I has been extensively investigated by flash-absorption spectroscopy in psaD and psaE deleted mutants from Synechocystis sp. PCC 6803. In both mutants, the dissociation constant for the photosystem I/ferredoxin complex at pH 8 is considerably increased as compared to the wild type: approximately 25- and 100-fold increases are found for PsaD-less and PsaE-less photosystem I, respectively. However, at high ferredoxin concentrations, submicrosecond and microsecond kinetics of electron transfer similar to that observed in the wild type are present in both mutants. The presence of these fast kinetic components indicates that the relative positions of ferredoxin and of the terminal photosystem I acceptor are not significantly disturbed by the absence of either PsaD or PsaE. The second-order rate constant of ferredoxin reduction is lowered 10- and 2-fold for PsaD-less and PsaE-less photosystem I, respectively. Assuming a simple binding equilibrium between photosystem I and ferredoxin, PsaD appears to be important for the guiding of ferredoxin to its binding site (main effect on the association rate) whereas PsaE seems to control the photosystem I/ferredoxin complex lifetime (main effect on the dissociation rate). The properties of electron transfer from photosystem I to ferredoxin were also studied at pH 5. 8. In the psaE deleted mutant as in the wild type, the change of pH from 8 to 5.8 induces a 10-fold increase in affinity of ferredoxin for photosystem I. In the absence of PsaD, this pH effect is not observed, in favor of this subunit being mostly responsible for the low pH increased affinity