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In the green alga Chlamydomonas reinhardtii, hydrogen production is catalyzed via the [FeFe]-hydrogenases HydA1 and HydA2. The electrons required for the catalysis are transferred from ferredoxin (FDX) towards the hydrogenases. In the light, ferredoxin receives its electrons from photosystem I (PSI) so that H2 production becomes a fully light-driven process. HydA1 and HydA2 are highly O2 sensitive; consequently, the formation of H2 occurs mainly under anoxic conditions. Yet, photo-H2 production is tightly coupled to the efficiency of photosynthetic electron transport and linked to the photosynthetic control via the Cyt b6f complex, the control of electron transfer at the level of photosystem II (PSII) and the structural remodeling of photosystem I (PSI). These processes also determine the efficiency of linear (LEF) and cyclic electron flow (CEF). The latter is competitive with H2 photoproduction. Additionally, the CBB cycle competes with H2 photoproduction. Consequently, an in-depth understanding of light-driven H2 production via photosynthetic electron transfer and its competition with CO2 fixation is essential for improving photo-H2 production. At the same time, the smart design of photo-H2 production schemes and photo-H2 bioreactors are challenges for efficient up-scaling of light-driven photo-H2 production. [ABSTRACT FROM AUTHOR]

The cytochrome b6f complex (b6f) has been initially considered as the ferredoxin-plastoquinone reductase (FQR) during cyclic electron flow (CEF) with photosystem I that is inhibited by antimycin A (AA). The binding of AA to the b6f Qi-site is aggravated by heme-ci, which challenged the FQR function of b6f during CEF. Alternative models suggest that PROTON GRADIENT REGULATION5 (PGR5) is involved in a b6f-independent, AA-sensitive FQR. Here, we show in Chlamydomonas reinhardtii that the b6f is conditionally inhibited by AA in vivo and that the inhibition did not require PGR5. Instead, activation of the STT7 kinase upon anaerobic treatment induced the AA sensitivity of b6f which was absent from stt7-1. However, a lock in State 2 due to persisting phosphorylation in the phosphatase double mutant pph1;pbcp did not increase AA sensitivity of electron transfer. The latter required a redox poise, supporting the view that state transitions and CEF are not coercively coupled. This suggests that the b6f-interacting kinase is required for structure-function modulation of the Qi-site under CEF favoring conditions. We propose that PGR5 and STT7 independently sustain AA-sensitive FQR activity of the b6f. Accordingly, PGR5-mediated electron injection into an STT7-modulated Qi-site drives a Mitchellian Q cycle in CEF conditions. [ABSTRACT FROM AUTHOR]

Photosynthesis and respiration rely upon a proton gradient to produce ATP. In photosynthesis, the Respiratory Complex I homologue, Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. However, little is known about the PS-CI molecular mechanism and attempts to understand its function have previously been frustrated by its large size and high lability. Here, we overcome these challenges by pushing the limits in sample size and spectroscopic sensitivity, to determine arguably the most important property of any electron transport enzyme – the reduction potentials of its cofactors, in this case the iron-sulphur clusters of PS-CI (N0, N1 and N2), and unambiguously assign them to the structure using double electron-electron resonance. We have thus determined the bioenergetics of the electron transfer relay and provide insight into the mechanism of PS-CI, laying the foundations for understanding of how this important bioenergetic complex functions. Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. Here the authors determine the reduction potentials of the iron-sulphur clusters of PS-CI and thus the bioenergetics of the electron transfer relay. [ABSTRACT FROM AUTHOR]

Most of life's energy comes from sunlight, and thus, photosynthesis underpins the survival of virtually all life forms. The light-driven electron transfer at photosystem I (PSI) is certainly the most important generator of reducing power at the cellular level and thereby largely determines the global amount of enthalpy in living systems (Nelson 2011). The PSI is a light-driven plastocyanin:ferredoxin oxidoreductase, which is embedded into thylakoid membranes of cyanobacteria and chloroplasts of eukaryotic photosynthetic organism. Structural determination of complexes of the photosynthetic machinery is vital for the understanding of its mode of action. Here, we describe new structural and functional insights into PSI and associated light-harvesting proteins, with a focus on the plasticity of PSI. [ABSTRACT FROM AUTHOR]

Photosystem I is defined as plastocyanin-ferredoxin oxidoreductase. Taking advantage of genetic engineering, kinetic analyses and cryo-EM, our data provide novel mechanistic insights into binding and electron transfer between PSI and Pc. Structural data at 2.74 Å resolution reveals strong hydrophobic interactions in the plant PSI-Pc ternary complex, leading to exclusion of water molecules from PsaA-PsaB/Pc interface once the PSI-Pc complex forms. Upon oxidation of Pc, a slight tilt of bound oxidized Pc allows water molecules to accommodate the space between Pc and PSI to drive Pc dissociation. Such a scenario is consistent with the six times larger dissociation constant of oxidized as compared with reduced Pc and mechanistically explains how this molecular machine optimized electron transfer for fast turnover. [ABSTRACT FROM AUTHOR]

Abstract: Eukaryotic photosystem I consists of two functional moieties: the photosystem I core, harboring the components for the light-driven charge separation and the subsequent electron transfer, and the peripheral light-harvesting complex (LHCI). While the photosystem I-core remained highly conserved throughout the evolution, with the exception of the oxidizing side of photosystem I, the LHCI complex shows a high degree of variability in size, subunits composition and bound pigments, which is due to the large variety of different habitats photosynthetic organisms dwell in. Besides summarizing the most current knowledge on the photosystem I-core structure, we will discuss the composition and structure of the LHCI complex from different eukaryotic organisms, both from the red and the green clade. Furthermore, mechanistic insights into electron transfer between the donor and acceptor side of photosystem I and its soluble electron transfer carrier proteins will be given. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts. [Copyright &y& Elsevier]

We used fast absorbance spectroscopy to investigate in vivo binding dynamics and electron transfer between plastocyanin (pc) and photosystem I (PSI), and cytochrome (cyt) f oxidation kinetics in Chlamydomonas reinhardtii mutants in which either the binding or the release of pc from PSI was diminished. Under single flash- excitation conditions, electron flow between PSI and the cyt complex was not affected by a 5-fold lowering of the binding affinity of pc to PSI as induced by a mutation replacing the tryptophan-651 of the PsaA subunit by a serine residue (PsaA- W651S). On the other hand, electron flow from PSI to the cyt b6f complex was very sensitive to a 2- to 3-fold decrease in the rate of pc release from PSI, obtained by replacing the glutamic acid residue 613 of the PsaB subunit with glutamine (PsaB-E613N). Thus, our data indicate that under these experimental conditions the release of oxidized pc limits electron transfer between cyt b6f complex and PSI in vivo. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying the onset of Fe-deficiency chlorosis and the maintenance of photosynthetic function in chlorotic chloroplasts are relevant to global photosynthetic productivity. We describe a series of graded responses of the photosynthetic apparatus to Fe-deficiency, including a novel response that occurs prior to the onset of chlorosis, namely the disconnection of the LHCI antenna from photosystem I (PSI). We propose that disconnection is mediated by a change in the physical properties of PSI-K in PSI in response to a change in plastid Fe con- tent, which is sensed through the occupancy, and hence activity, of the Fe-containing active site in Crdl. We show further that progression of the response involves remodeling of the antenna complexes-specific degradation of existing proteins coupled to the synthesis of new ones, and establishment of a new steady state with decreased stoichiometry of electron transfer complexes. We suggest that these responses are typical of a dynamic photosynthetic apparatus where photosynthetic function is optimized and photooxidative damage is minimized in graduated responses to a combination of nutrients, light quantity and quality. [ABSTRACT FROM AUTHOR]

Examines the fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii requires PsaF. PsaF-deficient mutant; Flash absorption spectroscopy; First-order kinetics among photosynthetic organisms.

Describes the binding characteristics and electron transfer between plastocyanin and photosystem I. Dissociation constants; Equilibrium of complex formation between reduced plastocyanin and photosystem I; Driving force and equilibrium of electron transfer.

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]

Under anaerobic conditions, several species of green algae perform a light-dependent hydrogen production catalyzed by a special group of [FeFe] hydrogenases termed HydA. Although highly interesting for biotechnological applications, the direct connection between photosynthetic electron transport and hydrogenase activity is still a matter of speculation. By establishing an in vitro reconstitution system, we demonstrate that the photosynthetic ferredoxin (PetF) is essential for efficient electron transfer between photosystem I and HydA1. To investigate the electrostatic interaction process and electron transfer between PetF and HydA1, we performed site-directed mutagenesis. Kinetic analyses with several site-directed mutagenesis variants of HydA1 and PetF enabled us to localize the respective contact sites. These experiments in combination with in silico docking analyses indicate that electrostatic interactions between the conserved HydA1 residue Lys396 and the C terminus of PetF as well as between the PetF residue Glu122 and the N-terminal amino group of HydA1 play a major role in complex formation and electron transfer. Mapping of relevant HydA1 and PetF residues constitutes an important basis for manipulating the physiological photosynthetic electron flow in favor of light-driven H2 production. [ABSTRACT FROM AUTHOR]

• CrNTRC can donate electrons to 2-Cys Peroxiredoxin1 in vitro. • CrNTRC reductase domain is characterized by a peculiar conformation, which was previously only described in Methanosarcina mazei. • The reductase domain of C136S mutant of CrNTRC is in the F O conformation. In plant chloroplasts, thiol regulation is driven by two systems. One relies on the activity of thioredoxins through their light dependent reduction by ferredoxin via a ferredoxin-thioredoxin reductase (FTR). In the other system, a NADPH-dependent redox regulation is driven by a NADPH-thioredoxin reductase C (NTRC). While the thioredoxin system has been deeply studied, a more thorough understanding of the function of this plant specific NTRC is desirable. NTRC is a single polypeptide harbouring a thioredoxin domain (Trx) at the C-terminus of a NADPH-dependent Thioredoxin reductase (TrxR). To provide functional and structural insights, we studied the crystal structure of the TrxR domain of the NTRC from Chlamydomonas reinhardtii (CrNTRC, Cre01.g054150.t1.2) and its Cys136Ser (C136S) mutant, which is characterized by the mutation of the resolving cysteine in the active site of the TrxR domain. Furthermore, we confirmed the role of NTRC as electron donor for 2-Cys peroxiredoxin (PRX) also in C. reinhardtii. The structural data of TrxR were employed to develop a scheme of action which addresses electron transfer between TrxR and Trx of NTRC and between NTRC and its substrates. [ABSTRACT FROM AUTHOR]

The binding and electron transfer between plastocyanin (pc) or cytochrome c6 (cyt c6) and photosystem I (PSI) can be described by hydrophic as well as electrostatic interactions. The two α helices, l and l' in PsaB and PsaA, respectively, are involved in forming the hydrophobic interaction site at the oxidizing site of PSI. To obtain mechanistic insights into the function of the two negatively charged residues D612 and E613, present in α helix l of PsaB, we exchanged both residues by site-directed mutagenesis with His and transformed a PsaB deficient mutant of Chlamydomonas reinhardtii. Flash-induced absorption spectroscopy revealed that PSI harboring the changes D612H and E613H had a high affinity toward binding ol the electron donors and possessed an altered pH dependence of electron transfer with pc and cyt c6. Despite optimized binding and electron transfer between the altered PSI and its electron donors, the mutant strain PsaB-D612H/E613H exhibited a strong light sensitive growth phenotype, indicating that decelerated turnover between pc/cyt c6 and PSI with respect to electron transfer is deleterious to the cells. [ABSTRACT FROM AUTHOR]

PGRL1 RNA and protein levels are increased in iron-deficient Chlamydomonas reinhardtii cells. In an RNAi strain, which accumulates lower PGRL1 levels in both iron-replete and -starved conditions, the photosynthetic electron transfer rate is decreased, respiratory capacity in iron-sufficient conditions is increased, and the efficiency of cyclic electron transfer under iron-deprivation is diminished. Pgrl1-kd cells exhibit iron deficiency symptoms at higher iron concentrations than wild-type cells, although the cells are not more depleted in cellular iron relative to wild-type cells as measured by mass spectrometry. Thiol-trapping experiments indicate iron-dependent and redox-induced conformational changes in PGRL1 that may provide a link between iron metabolism and the partitioning of photosynthetic electron transfer between linear and cyclic flow. We propose, therefore, that PGRL1 in C. reinhardtii may possess a dual function in the chloroplast; that is, iron sensing and modulation of electron transfer. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms are frequently exposed to excess light conditions and hence to photo-oxidative stress. To counteract photo-oxidative damage, land plants and most algae make use of non- photochemical quenching (NPQ) of excess light energy, in particular the rapidly inducible and relaxing qE-mechanism. In vascular plants, the constitutively active PsbS protein is the key regulator of qE. In the green algae C. reinhardtii , however, qE activation is only possible after initial high-light (HL) acclimation for several hours and requires the synthesis of LHCSR proteins which act as qE regulators. The precise function of PsbS, which is transiently expressed during HL acclimation in C. reinhardtii , is still unclear. Here, we investigated the impact of different PsbS amounts on HL acclimation characteristics of C. reinhardtii cells. We demonstrate that lower PsbS amounts negatively affect HL acclimation at different levels, including NPQ capacity, electron transport characteristics, antenna organization and morphological changes, resulting in an overall increased HL sensitivity and lower vitality of cells. Contrarily, higher PsbS amounts do not result in a higher NPQ capacity, but nevertheless provide higher fitness and tolerance towards HL stress. Strikingly, constitutively expressed PsbS protein was found to be degraded during HL acclimation. We propose that PsbS is transiently required during HL acclimation for the reorganization of thylakoid membranes and/or antenna proteins along with the activation of NPQ and adjustment of electron transfer characteristics, and that degradation of PsbS is essential in the fully HL acclimated state. • Reduced amount of PsbS negatively affects high light acclimation. • Increased amount of PsbS provides higher fitness and tolerance towards high light. • Increased amount of PsbS does not increase the maximum NPQ capacity. • Altered PsbS amounts retard the accumulation of LHCSR3. • Constitutively over-expressed PsbS protein is degraded upon high light acclimation. [ABSTRACT FROM AUTHOR]