Search

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 [2Fe2S] 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 [2Fe2S] 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Photosystem I (PSI), a large protein complex located in the thylakoid membrane, mediates the final step in light-driven electron transfer to the stromal electron carrier protein ferredoxin (Fd). Here, we report the first structural description of the PSI-Fd complex from Thermosynechococcus elongatus. The trimeric PSI complex binds three Fds in a non-equivalent manner. While each is recognized by a PSI protomer in a similar orientation, the distances between Fds and the PSI redox centres differ. Fd binding thus entails loss of the exact three-fold symmetry of the PSI's soluble subunits, inducing structural perturbations which are transferred to the lumen through PsaF. Affinity chromatography and nuclear magnetic resonance analyses of PSI-Fd complexes support the existence of two different Fd-binding states, with one Fd being more tightly bound than the others. We propose a dynamic structural basis for productive complex formation, which supports fast electron transfer between PSI and Fd.

Reduction of ferredoxin by photosystem I (PSI) involves the [4Fe-4S] clusters F A and F B harbored by PsaC, with F B being the direct electron transfer partner of ferredoxin (Fd). Binding of the redox-inactive gallium ferredoxin to PSI was investigated by flash-absorption spectroscopy, studying both the P700 + decay and the reduction of the native iron Fd in the presence of Fd Ga . Fd Ga binding resulted in a faster recombination between P700 + and (F A , F B ) - , a slower electron escape from (F A , F B ) - to exogenous acceptors, and a decreased amount of intracomplex Fd Fe reduction, in accordance with competitive binding between Fd Fe and Fd Ga . [Fd Ga ] titrations of these effects revealed that the dissociation constant for the PSI:Fd Ga complex is different whether (F A , F B ) is oxidized or singly reduced. This difference in binding, together with the increase in the recombination rate, could both be attributed to a c. -30 mV shift of the midpoint potential of (F A , F B ), considered as a single electron acceptor, due to Fd Ga binding. This effect of Fd Ga binding, which can be extrapolated to Fd Fe because of the highly similar structure and the identical charge of the two Fds, should help irreversibility of electron transfer within the PSI:Fd complex. The effect of Fd binding on the individual midpoint potentials of F A and F B is also discussed with respect to the possible consequences on intra-PSI electron transfer and on the escape process.

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 (F A F B ). Newly obtained values were derived firstly from the fact that the dissociation constant between PSI and redox-inactive gallium-substituted Fd increases upon (F A F B ) 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 (F A F B ) single reduction and by about one order of magnitude after electron transfer from (F A F B ) - to Fd, therefore favoring turnover of Fd reduction by PSI. With Thermosynechococcus elongatus partners, values of 270, 730 and >10000s -1 were thus determined for (F A F B )Fd oxidized , (F A F B ) - Fd oxidized and (F A F B )Fd reduced , 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 (< -60mV for Thermosynechococcus elongatus). From the present state of knowledge, the question is still open whether this redox shift is compatible with a large (>10) equilibrium constant for intracomplex reduction of Fd from (F A F B ) - ., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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. 6500s(-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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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 (FNR(ox))-NADPH and reduced FNR (FNR(red))-NADP(+), both having CT absorption bands centered at approximately 600n m. After mixing BsFNR(ox) with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNR(red) was mixed with NADP(+), the amount of BsFNR(ox) increased with increasing NADP(+) concentration, but BsFNR(red) 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 BsFd(red) with BsFNR(ox) 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 BsFd(red) increased from 40 to 120 μM. The reduction rate of BsFNR(ox) by BsFd(red) was markedly decreased by premixing BsFNR(ox) with BsFd(ox), indicating that the dissociation of BsFd(ox) from BsFNR(sq) is rate-limiting in the reaction. The characteristics of the BsFNR reactions with NADP(+)/NADPH were compared with those of other types of FNRs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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 × 10(7) 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.

The dissociation constant K(d) 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. K(d) values of 0.14-0.38 microM 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.

The electron transfer cascade from photosystem I to NADP+ was studied at physiological pH by flash-absorption spectroscopy in a Synechocystis PCC6803 reconstituted system comprised of purified photosystem I, ferredoxin, and ferredoxin-NADP+ reductase. Experiments were conducted with a 34-kDa ferredoxin-NADP+ reductase homologous to the chloroplast enzyme and a 38-kDa N-terminal extended form. Small differences in kinetic and catalytic properties were found for these two forms, although the largest one has a 3-fold decreased affinity for ferredoxin. The dissociation rate of reduced ferredoxin from photosystem I (800 s(-1)) and the redox potential of the first reduction of ferredoxin-NADP+ reductase (-380 mV) were determined. In the absence of NADP+, differential absorption spectra support the existence of a high affinity complex between oxidized ferredoxin and semireduced ferredoxin-NADP+ reductase. An effective rate of 140-170 s(-1) was also measured for the second reduction of ferredoxin-NADP+ reductase, this process having a rate constant similar to that of the first reduction. In the presence of NADP+, the second-order rate constant for the first reduction of ferredoxin-NADP+ reductase was 20% slower than in its absence, in line with the existence of ternary complexes (ferredoxin-NADP+ reductase)-NADP+-ferredoxin. A single catalytic turnover was monitored, with 50% NADP+ being reduced in 8-10 ms using 1.6 microM photosystem I. In conditions of multiple turnover, we determined initial rates of 360-410 electrons per s and per ferredox-in-NADP+ reductase for the reoxidation of 3.5 microM photoreduced ferredoxin. Identical rates were found with photosystem I lacking the PsaE subunit and wild type photosystem I. This suggests that, in contrast with previous proposals, the PsaE subunit is not involved in NADP+ photoreduction.

Nitrite reductases found in plants, algae, and cyanobacteria catalyze the six-electron reduction of nitrite to ammonia with reduced ferredoxin serving as the electron donor. They contain one siroheme and one [4Fe-4S] cluster, acting as separate one-electron carriers. Nitrite is thought to bind to the siroheme and to remain bound until its complete reduction to ammonia. In the present work the enzyme catalytic cycle, with ferredoxin reduced by photosystem 1 as an electron donor, has been studied by EPR and laser flash absorption spectroscopy. Substrate depletion during enzyme turnover, driven by a series of laser flashes, has been demonstrated. A complex of ferrous siroheme with NO, formed by two-electron reduction of the enzyme complex with nitrite, has been shown to be an intermediate in the enzyme catalytic cycle. The same complex can be formed by incubation of free oxidized nitrite reductase with an excess of nitrite and ascorbate. Hydroxylamine, another putative intermediate in the reduction of nitrite catalyzed by nitrite reductase, was found to react with oxidized nitrite reductase to produce the same ferrous siroheme-NO complex, with a characteristic formation time of about 13 min. The rate-limiting step for this reaction is probably hydroxylamine binding to the enzyme, with the conversion of hydroxylamine to NO at the enzyme active site likely being much faster.

This paper reports on extensive molecular dynamics simulations (about 40 ns in total) in both the reduced and the oxidized states of Ferredoxin from Cyanobacterium Anabaena PCC7119. These calculations have provided us with the free energy profile of the phi(47) backbone angle which controls the "CO in" to "CO out" transition of Cys46 in the reduced and oxidized Fd7119. Our main motivation has been to identify the time scales involved in the reduction of Fd and single out the amino acid residues crucially affecting the conformational change and, thus, electron transfer. The free energy profiles obtained in this study are relevant to electron transfers in the PSI/Fd7119 and Fd7119/FNR complexes. Our findings based on hydrated ferredoxin simulations are that activated processes are to occur in the protein during electron transfer to and from ferredoxin. The relative stability and the activation barrier of the "CO in" to "CO out" transition can be modulated by the distance between the Ser47 and the Glu94 residues. In our calculations, for short distances, the "CO in" state is favored in the reduced form, whereas for large distances, the "CO out" state becomes increasingly favored. Accordingly, conformational changes in Fd7119 when bound to PSI or FNR can have crucial effects on the kinetics of the electron transfer. Our simulations also show that the hydrogen bond between between Ser47(OG) and Cys46(O) is essential to lock in the "CO out" state. This finding explains why only the Ser47Thr Fd7119 mutant sustains electron transfer activity, as only residues serine and threonine can form a specific hydrogen bond with Cys46(O). Finally, our simulations predict that Phe65 has a large probability of being in close contact with the Cys46(O) at the top of the conformational free energy barrier. This carbonyl/phenyl ring interaction can then facilitate the de-localization of the Fd's electron toward the Pi orbitals of Phe65 aromatic ring which is thought to be crucial to the Fd7119/FNR electron transfer

The formation of a transient complex between photosystem I and ferredoxin is involved in the process of ferredoxin photoreduction in oxygenic photosynthetic organisms. Reduced ferredoxin is an essential redox intermediate involved in many assimilatory processes and is necessary for the reduction of NADP(+) to NADPH. Single crystals from a complex of photosystem I with ferredoxin were grown using PEG 400 and CaCl(2) as precipitation agents. The crystals diffract x-rays to a resolution of 7-8 A. The space group was determined to be orthorhombic with the unit cell dimensions a = 194 A, b = 208 A, and c = 354 A. The crystals contain photosystem I and ferredoxin in a 1:1 ratio. Electron paramagnetic resonance (EPR) measurements on these crystals are reported, where EPR signals of the three [4Fe-4S] clusters F(A), F(B), F(X), and the [2Fe-2S] cluster of ferredoxin were detected. From the EPR spectra observed at three particular orientations of the crystal in the magnetic field, the full orientation pattern of the F g-tensor was simulated. This simulation is consistent with the presence of 12 magnetically inequivalent F clusters per unit cell with the C(3) axis of the PSI trimers oriented at (23 degrees, 72 degrees, 77 degrees ) to the unit cell axes.

The reaction center of photosystem I (PSI) reduces soluble ferredoxin on the stromal side of the photosynthetic membranes of cyanobacteria and chloroplasts. The X-ray structure of PSI from the cyanobacterium Synechococcus elongatus has been recently established at a 2.5 A resolution [Nature 411 (2001) 909]. The kinetics of ferredoxin photoreduction has been studied in recent years in many mutants of the stromal subunits PsaC, PsaD and PsaE of PSI. We discuss the ferredoxin docking site of PSI using the X-ray structure and the effects brought by the PSI mutations to the ferredoxin affinity.

Abstract: We measured the kinetics of light-induced NADPH formation and subsequent dark consumption by monitoring in vivo its fluorescence in the cyanobacterium Synechocystis PCC 6803. Spectral data allowed the signal changes to be attributed to NAD(P)H and signal linearity vs the chlorophyll concentration was shown to be recoverable after appropriate correction. Parameters associated to reduction of NADP+ to NADPH by ferredoxin–NADP+-oxidoreductase were determined: After single excitation of photosystem I, half of the signal rise is observed in 8ms; Evidence for a kinetic limitation which is attributed to an enzyme bottleneck is provided; After two closely separated saturating flashes eliciting two photosystem I turnovers in less than 2ms, more than 50% of the cytoplasmic photoreductants (reduced ferredoxin and photosystem I acceptors) are diverted from NADPH formation by competing processes. Signal quantitation in absolute NADPH concentrations was performed by adding exogenous NADPH to the cell suspensions and by estimating the enhancement factor of in vivo fluorescence (between 2 and 4). The size of the visible (light-dependent) NADP (NADP+ +NADPH) pool was measured to be between 1.4 and 4 times the photosystem I concentration. A quantitative discrepancy is found between net oxygen evolution and NADPH consumption by the light-activated Calvin–Benson cycle. The present study shows that NADPH fluorescence is an efficient probe for studying in vivo the energetic metabolism of cyanobacteria which can be used for assessing multiple phenomena occurring over different time scales. [Copyright &y& Elsevier]

Flavodiiron proteins (FDPs) of photosynthetic organisms play a photoprotective role by reducing oxygen to water and thus avoiding the accumulation of excess electrons on the photosystem I (PSI) acceptor side under stress conditions. In Synechocystis sp. PCC 6803 grown under high CO 2 , both FDPs Flv1 and Flv3 are indispensable for oxygen reduction. We performed a detailed in vivo kinetic study of wild-type (WT) and Δ flv1/3 strains of Synechocystis using light-induced NADPH fluorescence and near-infrared absorption of iron-sulfur clusters from ferredoxin and the PSI acceptors (F A F B), collectively named FeS. These measurements were performed under conditions where the Calvin-Benson cycle is inactive or poorly activated. Under such conditions, the NADPH decay following a short illumination decays in parallel in both strains and exhibits a time lag which is correlated to the presence of reduced FeS. On the contrary, reduced FeS decays much faster in WT than in Δ flv1/3 (13 vs 2 s−1). These data unambiguously show that reduced ferredoxin, or possibly reduced F A F B , is the direct electron donor to the Flv1/Flv3 heterodimer. Evidences for large reduction of (F A F B) and recombination reactions within PSI were also provided by near-infrared absorption. Mutants lacking either the NDH1-L complex, the homolog of complex I of respiration, or the Pgr5 protein show no difference with WT in the oxidation of reduced FeS following a short illumination. These observations question the participation of a significant cyclic electron flow in cyanobacteria during the first seconds of the induction phase of photosynthesis. • Ferredoxin is most likely the electron donor to flavodiiron proteins in Synechocystis. • The terminal photosystem I acceptor cannot be excluded as the electron donor. • Recombination reactions are observed in vivo with near-infrared absorption. • Cyclic electron flow is not significant during the first seconds of illumination. [ABSTRACT FROM AUTHOR]