Search

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 > 10000 s −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 (< −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 (> 10) equilibrium constant for intracomplex reduction of Fd from (F A F B ) − . [ABSTRACT FROM AUTHOR]

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]

The phycobilisome, the cyanobacterial light harvesting complex, is a huge phycobiliprotein containing extramembrane complex, formed by a core from which rods radiate. The phycobilisome has evolved to efficiently absorb sun energy and transfer it to the photosystems via the last energy acceptors of the phycobilisome, ApcD and ApcE. ApcF also affects energy transfer by interacting with ApcE. In this work we studied the role of ApcD and ApcF in energy transfer and state transitions in Synechococcus elongatus and Synechocystis PCC6803. Our results demonstrate that these proteins have different roles in both processes in the two strains. The lack of ApcD and ApcF inhibits state transitions in Synechocystis but not in S. elongatus. In addition, lack of ApcF decreases energy transfer to both photosystems only in Synechocystis , while the lack of ApcD alters energy transfer to photosystem I only in S. elongatus. Thus, conclusions based on results obtained in one cyanobacterial strain cannot be systematically transferred to other strains and the putative role(s) of phycobilisomes in state transitions need to be reconsidered. Unlabelled Image • ApcD deletion in S. elongatus does not impair state transitions. • Lack of ApcD decreases energy transfer from PBSs to PSI in S. elongatus. • ApcF deletion decreases energy transfer from PBSs to PSII and PSI in Synechocystis. • PBS involvement in state transition might differ in S. elongatus and Synechocystis. [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]

Leaves of Arabidopsis thaliana plants grown in short days (8 h light) generate more reactive oxygen species in the light than leaves of plants grown in long days (16 h light). The importance of the two PsaE isoforms of photosystem I, PsaE1 and PsaE2, for O 2 reduction was studied in plants grown under these different growth regimes. In short day conditions a mutant affected in the amount of PsaE1 (psae1-1) reduced more efficiently O 2 than a mutant lacking PsaE2 (psae2-1) as shown by spin trapping EPR spectroscopy on leaves and by following the kinetics of P700+ reduction in isolated photosystem I. In short day conditions higher O 2 reduction protected photosystem II against photoinhibition in psae1-1. In contrast in long day conditions the presence of PsaE1 was clearly beneficial for photosynthetic electron transport and for the stability of the photosynthetic apparatus under photoinhibitory conditions. We conclude that the two PsaE isoforms have distinct functions and we propose that O 2 reduction at photosystem I is beneficial for the plant under certain environmental conditions. • Plants grown in short day generate more ROS at photosystem I than in long day. • The subunit PsaE2 of photosystem I facilitates the reduction of oxygen. • Reduction of oxygen at PSI lowers the susceptibility of PSII to photoinhibition. [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]