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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]

Nitrite reductase, which reduces nitrite to ammonium in a six-electron reaction, was characterized through kinetic analysis of an electron transfer cascade involving photoexcited Photosystem I and ferredoxin. This cascade was studied at physiological pH by flash-absorption spectroscopy. Two different forms of the enzyme were studied: one isolated from spinach leaf and one histidine-tagged recombinant form. When the enzyme is oxidized in the absence of nitrite, single-enzyme reduction leads mostly to siroheme reduction with the leaf enzyme, whereas the siroheme and the [4Fe-4S} cluster are both reduced in equivalent amounts in the recombinant enzyme. When combined with the results of deazaflavin/EDTA photoreduction experiments, these data support a 50 mV negative shift of the siroheme midpoint potential in the recombinant enzyme. Despite this difference, the two forms of the enzyme exhibit similar values for the rate constant of single reduction by reduced ferredoxin (1200 s-1) and for kcat (420-450 electrons per second and per nitrite reductase). When nitrite reductase is initially pre-reduced to the state ferrous siroheme-NO., the fast kinetics of reduction by ferredoxin and the thermodynamics of ferredoxin binding are equivalent to those observed with oxidized nitrite reductase without nitrite. Spectral and kinetic analyses of single reduction of the recombinant enzyme in the ferrous siroheme-NO. state by photoreduced ferredoxin reveal that this process leads to reduction of the [4Fe-4S] cluster with little, if any, NO. reduction. These data show that the enzyme must wait for the next reduction step before NO. undergoes substantial reduction. [ABSTRACT FROM AUTHOR]

PsaC is the stromal subunit of photosystem I (PSI) which binds the two terminal electron acceptors FA and FB. This subunit resembles 2[4Fe-4S] bacterial ferredoxins but contains two additional sequences: an internal loop and a C-terminal extension. To gain new insights into the function of the internal loop, we used an in vivo degenerate oligonucleotide-directed mutagenesis approach for analysing this region in the green alga Chlamydomonas reinhardtii. Analysis of several psaC mutants affected in PSI function or assembly revealed that K35 is a main interaction site between PsaC and ferredoxin (Fd) and that it plays a key role in the electrostatic interaction between Fd and PSI. This is based upon the observation that the mutations K35T, K35D and K35E drastically affect electron transfer from PSI to Fd, as measured by flash-absorption spectroscopy, whereas the K35R change has no effect on Fd reduction. Chemical cross-linking experiments show that Fd interacts not only with PsaD and PsaE, but also with the PsaC subunit of PSI. Replacement of K35 by T, D, E or R abolishes Fd cross-linking to PsaC, and cross-linking to PsaD and PsaE is reduced in the K35T, K35D and K35E mutants. In contrast, replacement of any other lysine of PsaC does not alter the cross-linking pattern, thus indicating that K35 is an interaction site between PsaC and its redox partner Fd. [ABSTRACT FROM AUTHOR]

The dissociation constant κd of the photosystem I (PSI): ferredoxin complex has been measured by backscattering interferometry (BSI) with cyanobacterial PSI (350 kDa) and ferredoxin (10.5 kDa). The BSI signal, consisting of shifts for interference fringes resulting from a change in refractive index due to complex formation, was monitored as ferredoxin concentration was titrated. κd values of 0.14-0.38 μM were obtained with wild-type PSI whereas no complex was detectable with a PSI mutant containing a single mutation (R39Q) in the PsaE extrinsic subunit. These results are in quantitative agreement with previous functional determinations consisting in the detection of fast electron transfer within the complex. They provide evidence that the main contribution for the high affinity binding of ferredoxin to PSI is due to a single region of PsaE comprising arginine 39. They do not support the existence of a secondary binding site that could have escaped functional detection. [ABSTRACT FROM AUTHOR]

A flash photolysis study of electron transfer (ET) kinetics from reduced ferredoxin (photoreduced by Photosystem I) to the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 has been carried out. In the presence of nitrate, under conditions where only a single electron is transferred to nitrate reductase, the rate of enzyme reduction shows a biphasic concentration dependence: At low enzyme concentrations the dependence is approximately linear, with an estimated second-order rate constant of 7.4 ± 0.8 x 107 M-1 s-1; at concentrations above 2 µM, the rate increases nonlinearly to an asymptotic value of approximately 300 s-1, indicating the presence of a rate-limiting step in the process. The spectrum of the one-electron reduced enzyme suggests that Mo centers are largely reduced with a minor contribution of iron--sulfur cluster reduction. Under conditions favoring two-electron reduction of the enzyme, the redox difference spectrum can be accounted for by the oxidation of two reduced ferredoxins, suggesting that the enzyme has completed one full catalytic cycle. The spectral changes observed in the absence of nitrate are significantly different from those seen in the presence of nitrate. Experiments in the absence of nitrate revealed that the singly reduced enzyme exhibits different absorption characteristics and reoxidation kinetics, compared to those observed with nitrate present, and exhibits a much faster binding by reduced ferredoxin than the oxidized enzyme. The implications of these observations for understanding the enzyme mechanism are discussed. [ABSTRACT FROM AUTHOR]

Photosystem I (PSI) is an essential protein complex for oxygenic photosynthesis and is also known to be an important source of reactive oxygen species (ROS) in the light. When ROS are generated within PSI, the photosystem can be damaged. The so-called PSI photoinhibition is a lethal event for oxygenic phototrophs, and it is prevented by keeping the reaction center chlorophyll (P700) oxidized in excess light conditions. Whereas regulatory mechanisms for controlling P700 oxidation have been discovered already, the molecular mechanism of PSI photoinhibition is still unclear. Here, we characterized the damage mechanism of PSI photoinhibition by in vitro transient absorption and electron paramagnetic resonance (EPR) spectroscopy in isolated PSI from cucumber leaves that had been subjected to photoinhibition treatment. Photodamage to PSI was induced by two different light treatments: 1. continuous illumination with high light at low (chilling) temperature (C/LT) and 2. repetitive flashes at room temperature (F/RT). These samples were compared to samples that had been illuminated with high light at room temperature (C/RT). The [Fe S] clusters F X and (F A F B) were destructed in C/LT but not in F/RT. Transient absorption spectroscopy indicated that half of the charge separation was impaired in F/RT, however, low-temperature EPR revealed the light-induced F X signal at a similar size as in the case of C/RT. This indicates that the two branches of electron transfer in PSI were affected differently. Electron transfer at the A-branch was inhibited in F/RT and also partially in C/LT, while the B-branch remained active. • Photosystem I photoinhibition differs between different illumination protocols. • Fe-S clusters are destroyed by illumination at chilling temperature. • The A- and B-branches in photosystem I are affected differently by photoinhibition. [ABSTRACT FROM AUTHOR]

A multi-enzyme electron-transfer chain involving solubilized photosystem I (PSI) as photocatalytic unit, cytochrome c6 and ferredoxin as electron carriers and ferredoxin/NADPH oxidoreductase (FNR) as electron acceptor was reconstituted in an electrochemical cell and studied by cyclic voltammetry. The working gold electrodes were modified to react selectively with cytochrome C6. Quantitative analysis of the photocatalytic current under continuous illumination allowed the determination of the values kon and koff for the ferredoxin/PSI interaction. An efficient recycling system for NADPH was established, and the dissociation constant of the oxidized ferredoxin/semiquinone FNR complex was extracted by modeling the catalytic efficiency of the chain as a function of ferredoxin concentration. The value determined hereby is consistent with a shift of -50 to -100 mV of the reduction potential of ferredoxin when complexed with FNR. [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 complex I enables cyclic electron flow around photosystem I, a regulatory mechanism for photosynthetic energy conversion. We report a 3.3-angstrom-resolution cryo–electron microscopy structure of photosynthetic complex I from the cyanobacterium Thermosynechococcus elongatus. The model reveals structural adaptations that facilitate binding and electron transfer from the photosynthetic electron carrier ferredoxin. By mimicking cyclic electron flow with isolated components in vitro, we demonstrate that ferredoxin directly mediates electron transfer between photosystem I and complex I, instead of using intermediates such as NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate). A large rate constant for association of ferredoxin to complex I indicates efficient recognition, with the protein subunit NdhS being the key component in this process. [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]