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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]

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]

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]

Illumination of photosystem I (PSI) from the cyanobacterium Synechocystis sp. PCC 6803 at 200 K in the presence of ascorbate leads to the formation of two ascorbyl radicals per PSI, which are formed by P700+ reduction by ascorbate. During photoaccumulation, one half of the ascorbyl radicals is formed with a halftime of 1 min and the other half with a halftime of 7 min. Pulsed electron paramagnetic resonance (EPR) experiments with protonated/deuterated PSI show that a PSI proton/deuteron is strongly coupled to the ascorbyl radical. Our data indicate that reactive ascorbate molecules bind to PSI at two specific locations, which might be symmetrically located with respect to the pseudo-C2 axis of symmetry of the heterodimeric core of PSI. Reduction of P700+ by ascorbate leads to multiple turnover of PSI photochemistry, resulting in partial photoaccumulation of the doubly reduced species (FA-, FB-). A modified form of FB-—in accordance with Chamorovsky and Cammack [Biochim. Biophys. Acta 679 (1982) 146–155], but not of FA-, is observed by EPR after illumination at 200 K, which indicates that reduction of FB at 200 K is followed by some relaxation process, in line with this cluster being the most exposed to the solvent. [Copyright &y& Elsevier]

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. [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]

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]

Ferredoxin-NADP + oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis ( Bs FNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of Bs FNR with NADP + , NADPH, NADPD (deuterated form) and B. subtilis ferredoxin ( Bs Fd) using stopped-flow spectrophotometry were studied. Mixing Bs FNR 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 600 nm. After mixing Bs FNR ox with about a 10-fold molar excess of NADPH (forward reaction), Bs FNR was almost completely reduced at equilibrium. When Bs FNR red was mixed with NADP + , the amount of Bs FNR ox increased with increasing NADP + concentration, but Bs FNR 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 Bs Fd red with Bs FNR 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 Bs Fd red increased from 40 to 120 μM. The reduction rate of Bs FNR ox by Bs Fd red was markedly decreased by premixing Bs FNR ox with Bs Fd ox , indicating that the dissociation of Bs Fd ox from Bs FNR sq is rate-limiting in the reaction. The characteristics of the Bs FNR reactions with NADP + /NADPH were compared with those of other types of FNRs. [ABSTRACT FROM AUTHOR]

Biological desert sand crusts are the foundation of desert ecosystems, stabilizing the sands and allowing colonization by higher order organisms. The first colonizers of the desert sands are cyanobacteria. Facing the harsh conditions of the desert, these organisms must withstand frequent desiccation–hydration cycles, combined with high light intensities. Here, we characterize structural and functional modifications to the photosynthetic apparatus that enable a cyanobacterium, Leptolyngbya sp., to thrive under these conditions. Using multiple in vivo spectroscopic and imaging techniques, we identified two complementary mechanisms for dissipating absorbed energy in the desiccated state. The first mechanism involves the reorganization of the phycobilisome antenna system, increasing excitonic coupling between antenna components. This provides better energy dissipation in the antenna rather than directed exciton transfer to the reaction center. The second mechanism is driven by constriction of the thylakoid lumen which limits diffusion of plastocyanin to P 700 . The accumulation of P 700 + not only prevents light-induced charge separation but also efficiently quenches excitation energy. These protection mechanisms employ existing components of the photosynthetic apparatus, forming two distinct functional modes. Small changes in the structure of the thylakoid membranes are sufficient for quenching of all absorbed energy in the desiccated state, protecting the photosynthetic apparatus from photoinhibitory damage. These changes can be easily reversed upon rehydration, returning the system to its high photosynthetic quantum efficiency. [ABSTRACT FROM AUTHOR]

The plastid terminal oxidase PTOX catalyzes the oxidation of plastoquinol (PQH 2 ) coupled with the reduction of oxygen to water. In vivo PTOX is attached to the thylakoid membrane. PTOX is important for plastid development and carotenoid biosynthesis, and its role in photosynthesis is controversially discussed. To analyze PTOX activity in photosynthetic electron transport recombinant purified PTOX fused to the maltose-binding protein was added to photosystem II-enriched membrane fragments. These membrane fragments contain the plastoquinone (PQ) pool as verified by thermoluminescence. Experimental evidence for PTOX oxidizing PQH 2 is demonstrated by following chlorophyll fluorescence induction. Addition of PTOX to photosystem II-enriched membrane fragments led to a slower rise, a lower level of the maximal fluorescence and an acceleration of the fluorescence decay. This effect was only observed at low light intensities indicating that PTOX cannot compete efficiently with the reduction of the PQ pool by photosystem II at higher light intensities. PTOX attached tightly to the membranes since it was only partly removable by membrane washings. Divalent cations enhanced the effect of PTOX on chlorophyll fluorescence compared to NaCl most likely because they increase connectivity between photosystem II centers and the size of the PQ pool. Using single turnover flashes, it was shown that the level of reactive oxygen species, generated by PTOX in a side reaction, increased when the spacing between subsequent double flashes was enlarged. This shows that PTOX generates reactive oxygen species under limited substrate availability. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms need to sense and respond to fluctuating environmental conditions, to perform efficient photosynthesis and avoid the formation of harmful reactive oxygen species. Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome, the extramembranal light-harvesting antenna. This mechanism is triggered by the photoactive orange carotenoid protein (OCP). In this study, we characterized OCP and the related photoprotective mechanism in non-stressed and light-stressed cells of three different strains of Planktothrix that can form impressive blooms. In addition to changing lake ecosystemic functions and biodiversity, Planktothrix blooms can have adverse effects on human and animal health as they produce toxins (e.g., microcystins). Three Planktothrix strains were selected: two green strains, PCC 10110 (microcystin producer) and PCC 7805 (non-microcystin producer), and one red strain, PCC 7821. The green strains colonize shallow lakes with higher light intensities while red strains proliferate in deep lakes. Our study allowed us to conclude that there is a correlation between the ecological niche in which these strains proliferate and the rates of induction and recovery of OCP-related photoprotection. However, differences in the resistance to prolonged high-light stress were correlated to a better replacement of damaged D1 protein and not to differences in OCP photoprotection. Finally, microcystins do not seem to be involved in photoprotection as was previously suggested. Unlabelled Image • The Orange Carotenoid Protein related photoprotection in three Plankthothrix strains. • There is a correlation between ecological niche and OCP-related photoprotection. • Resistance to high-light was related to a better replacement of damaged D1. • Microcystins do not seem to be involved in photoprotection. [ABSTRACT FROM AUTHOR]