Search

We report the first in vivo analysis of a canonical CP12 regulatory protein, namely the unique CP12 of the model cyanobacterium Synechocystis PCC 6803, which has the advantage of being able to grow photoautotrophically, photomixotrophically, and photoheterotrophically. The data showed that CP12 is dispensable to cell growth under standard (continuous) light and light/dark cycle, whereas it is essential for the catabolism of exogenously added glucose that normally sustains cell growth in absence of photosynthesis. Furthermore, to be active in glucose catabolism, CP12 requires its three conserved features: its AWD_VEEL motif and its two pairs of cysteine residues. Also interestingly, CP12 was found to regulate the redox equilibrium of NADPH, an activity involving its AWD_VEEL motif and its C-ter cysteine residues, but not its N-ter cysteine residues. This finding is important because NADPH powers up the methylerythritol 4-phosphate (MEP) pathway that synthesizes the geranyl-diphosphate (GPP) and farnesyl-diphosphate (FPP) metabolites, which can be transformed into highvalue terpenes by recombinant cyanobacteria producing plant terpene synthase enzymes. Therefore, we have introduced into the Δcp12 mutant and the wildtype (control) strain our replicative plasmids directing the production of the monoterpene limonene and the sesquiterpene bisabolene. The photosynthetic production of both bisabolene and limonene appeared to be increased (more than two-fold) in the Δcp12 mutant as compared to the WT strain. Furthermore, the level of bisabolene production was also higher to those previously reported for various strains of Synechocystis PCC 6803 growing under standard (nonoptimized) photoautotrophic conditions. Hence, the presently described Δcp12 strain with a healthy photoautotrophic growth and an increased capability to produce terpenes, is an attractive cell chassis for further gene manipulations aiming at engineering cyanobacteria for high-level photoproduction of terpenes. [ABSTRACT FROM AUTHOR]

Summary: During daffodil flower development, chloroplasts differentiate into photosynthetically inactive chromoplasts having lost functional photosynthetic reaction centers. Chromoplasts exhibit a respiratory activity reducing oxygen to water and generating ATP. Immunoblots revealed the presence of the plastid terminal oxidase (PTOX), the NAD(P)H dehydrogenase (NDH) complex, the cytochrome b6f complex, ATP synthase and several isoforms of ferredoxin‐NADP+ oxidoreductase (FNR), and ferredoxin (Fd). Fluorescence spectroscopy allowed the detection of chlorophyll a in the cytochrome b6f complex. Here we characterize the electron transport pathway of chromorespiration by using specific inhibitors for the NDH complex, the cytochrome b6f complex, FNR and redox‐inactive Fd in which the iron was replaced by gallium. Our data suggest an electron flow via two separate pathways, both reducing plastoquinone (PQ) and using PTOX as oxidase. The first oxidizes NADPH via FNR, Fd and cytochrome bh of the cytochrome b6f complex, and does not result in the pumping of protons across the membrane. In the second, electron transport takes place via the NDH complex using both NADH and NADPH as electron donor. FNR and Fd are not involved in this pathway. The NDH complex is responsible for the generation of the proton gradient. We propose a model for chromorespiration that may also be relevant for the understanding of chlororespiration and for the characterization of the electron input from Fd to the cytochrome b6f complex during cyclic electron transport in chloroplasts. Significance Statement: Chromorespiration takes place via two pathways, one depends on FNR, ferredoxin, the cytochrome b6f complex, and the other depends on the NDH complex and is ferredoxin independent. We propose an electron transport via the cytochrome b6f complex that involves neither a Q‐cycle nor a high potential electron transport chain [ABSTRACT FROM AUTHOR]

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]

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]

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]

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 PCC71 19. These calculations have provided us with the free energy profile of the φ[SUB47] 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/Fd71 19 and Fd71 1 9/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 G1u94 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 Fd71 19 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. [ABSTRACT FROM AUTHOR]

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]

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]

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]

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]

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-l) 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 μM photosystem I. In conditions of multiple turnover, we determined initial rates of 360–410 electrons per s and per ferredoxin-NADP+ reductase for the reoxidation of 3.5 μM 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. [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]

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]

In photosynthetic organisms, ferredoxin:NADP+ oxidoreductase (FNR) is known to provide NADPH for CO2 assimilation, but it also utilizes NADPH to provide reduced ferredoxin. The cyanobacterium Synechocystis sp. strain PCC6803 produces two FNR isoforms, a small one (FNRS) similar to the one found in plant plastids and a large one (FNRL) that is associated with the phycobilisorne, a light-harvesting complex. Here we show that a mutant lacking FNRL exhibits a higher NADP+/NADPH ratio. We also purified to homogeneity a phycobilisome sub-complex comprising FNRL, named FNRL-PC. The enzymatic activities of FNRL-PC were compared with those of FNRs. During NADPH oxidation, FNRL-PC exhibits a 30% decrease in the Michaelis constant Km(NADPH), and a 70% increase in Km(ferredoxin), which is in agreement with its predicted lower activity of ferredoxin reduction. During NADP+ reduction, the FNRL-PC shows a 29/43% decrease in the rate of single electron transfer from reduced ferredoxin in the presence/absence of NADP+. The increase in Km(ferredoxin), and the rate decrease of single reduction are attributed to steric hindrance by the phycocyanin moiety of FNRL-PC. Both isoforms are capable of catalyzing the NADP+ reduction under multiple turnover conditions. Furthermore, we obtained evidence that, under high ionic strength conditions, electron transfer from reduced ferredoxin is rate limiting during this process. The differences that we observe might not fully explain the in vivo properties of the Synechocystis mutants expressing only one of the isoforms. Therefore, we advocate that FNR localization and/or substrates availability are essential in vivo. [ABSTRACT FROM AUTHOR]

A light-activated electron-transfer chain was assembled using solubilized cyanobacterialphotosystem I as photoactive enzyme, cytochrome 06 (also from cyanobacteria) as electron donor, andmethyl viologen as electron acceptor. The photocatalytic activity of the ensemble was measured by directand reversible electrochemistry of cytochrome 06 at a surface-modified gold electrode. Analysis of theelectrochemical response with an appropriate model for the reaction mechanism allowed the relation of the overall catalytic reaction rate to the individual steps of the catalytic cycle. Second-order rate constants were determined for the first time under steady-state conditions. The results validate this approach as an efficient method for the study of electron transfer between photoactive enzymes and their redox partners. [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]

Thiol-dependent redox regulation of enzyme activities plays a central role in regulating photosynthesis. Beside the regulation of metabolic pathways, alternative electron transport is subjected to thiol-dependent regulation. We investigated the regulation of O2 reduction at photosystem I. The level of O2 reduction in leaves and isolated thylakoid membranes depends on the photoperiod in which plants are grown. We used a set of Arabidopsis (Arabidopsis thaliana) mutant plants affected in the stromal, membrane and lumenal thiol network to study the redox protein partners involved in regulating O2 reduction. Light-dependent O2 reduction was determined in leaves and in thylakoids of plants grown in short day and long day conditions using a spin-trapping electron paramagnetic resonance (EPR) assay. In wild type samples from short day conditions, reactive oxygen species (ROS) generation was double that of samples from long day conditions, while this difference was abolished in several redoxin mutants. An in vitro reconstitution assay showed that thioredoxin m, NADPH-dependent reductase C and NADPH are required for high O2 reduction levels in thylakoids from plants grown in long day conditions. Using isolated photosystem I, we also showed that reduction of a photosystem I protein is responsible for the increase in O2 reduction. Furthermore, differences in the membrane localization of m-type thioredoxins and 2-Cys peroxiredoxin were detected between thylakoids of short day and long day plants. Overall, we propose a model of redox regulation of O2 reduction according to the reduction power of the stroma and the ability of different thiol-containing proteins to form a network of redox interactions., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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 organisms must sense and respond to fluctuating environmental conditions in order to perform efficient photosynthesis and to avoid the formation of dangerous reactive oxygen species. The excitation energy arriving at each photosystem permanently changes due to variations in the intensity and spectral properties of the absorbed light. Cyanobacteria, like plants and algae, have developed a mechanism, named "state transitions," that balances photosystem activities. Here, we characterize the role of the cytochrome b 6 f complex and phosphorylation reactions in cyanobacterial state transitions using Synechococcus elongatus PCC 7942 and Synechocystis PCC 6803 as model organisms. First, large photosystem II (PSII) fluorescence quenching was observed in State II, a result that does not appear to be related to energy transfer from PSII to PSI (spillover). This membrane-associated process was inhibited by betaine, Suc, and high concentrations of phosphate. Then, using different chemicals affecting the plastoquinone pool redox state and cytochrome b 6 f activity, we demonstrate that this complex is not involved in state transitions in S. elongatus or Synechocystis PCC6803. Finally, by constructing and characterizing 21 protein kinase and phosphatase mutants and using chemical inhibitors, we demonstrate that phosphorylation reactions are not essential for cyanobacterial state transitions. Thus, signal transduction is completely different in cyanobacterial and plant (green alga) state transitions., (© 2019 American Society of Plant Biologists. All rights reserved.)

In photosynthetic organisms, ferredoxin:NADP(+) oxidoreductase (FNR) is known to provide NADPH for CO(2) assimilation, but it also utilizes NADPH to provide reduced ferredoxin. The cyanobacterium Synechocystis sp. strain PCC6803 produces two FNR isoforms, a small one (FNR(S)) similar to the one found in plant plastids and a large one (FNR(L)) that is associated with the phycobilisome, a light-harvesting complex. Here we show that a mutant lacking FNR(L) exhibits a higher NADP(+)/NADPH ratio. We also purified to homogeneity a phycobilisome subcomplex comprising FNR(L,) named FNR(L)-PC. The enzymatic activities of FNR(L)-PC were compared with those of FNR(S). During NADPH oxidation, FNR(L)-PC exhibits a 30% decrease in the Michaelis constant K(m)((NADPH)), and a 70% increase in K(m)((ferredoxin)), which is in agreement with its predicted lower activity of ferredoxin reduction. During NADP(+) reduction, the FNR(L)-PC shows a 29/43% decrease in the rate of single electron transfer from reduced ferredoxin in the presence/absence of NADP(+). The increase in K(m)((ferredoxin)) and the rate decrease of single reduction are attributed to steric hindrance by the phycocyanin moiety of FNR(L)-PC. Both isoforms are capable of catalyzing the NADP(+) reduction under multiple turnover conditions. Furthermore, we obtained evidence that, under high ionic strength conditions, electron transfer from reduced ferredoxin is rate limiting during this process. The differences that we observe might not fully explain the in vivo properties of the Synechocystis mutants expressing only one of the isoforms. Therefore, we advocate that FNR localization and/or substrates availability are essential in vivo.

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.

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.