In Vivo Assembly of Photosystem I-Hydrogenase Chimera for In Vitro PhotoH2 Production

Funding Information: P.W., A.F., and J.A. contributed equally to this work. The authors are grateful to the Bundesministerium für Bildung und Forschung (BMBF) in the framework of the project CyFun (03SF0652A). The authors also thank Prof. Wolfgang Lubitz (Max Planck Institute for Chemical Energy Conversion, Mülheim an der Ruhr) for providing the DvMF[NiFe]-H2ase used for the fabrication of the H2 microsensor. Part of the project was funded by the research training group GRK2341 “Microbial Substrate Conversion (MiCon)” of the German research council (DFG) and the Dietmar Hopp Stiftung. P.W. is grateful for the financial support provided by the China Scholarship Council (CSC). F.C. is grateful to the support provided by FCT–Fundação para a Ciência e a Tecnologia, I.P. through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020). Open access funding enabled and organized by Projekt DEAL. Funding Information: P.W., A.F., and J.A. contributed equally to this work. The authors are grateful to the Bundesministerium für Bildung und Forschung (BMBF) in the framework of the project CyFun (03SF0652A). The authors also thank Prof. Wolfgang Lubitz (Max Planck Institute for Chemical Energy Conversion, Mülheim an der Ruhr) for providing the DvMF[NiFe]‐Hase used for the fabrication of the H microsensor. Part of the project was funded by the research training group GRK2341 “Microbial Substrate Conversion (MiCon)” of the German research council (DFG) and the Dietmar Hopp Stiftung. P.W. is grateful for the financial support provided by the China Scholarship Council (CSC). F.C. is grateful to the support provided by FCT–Fundação para a Ciência e a Tecnologia, I.P. through MOSTMICRO‐ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020). 2 2 Publisher Copyright: © 2023 The Authors. Advanced Energy Materials published by Wiley-VCH GmbH.Photosynthetic hydrogen (photoH2) production is an elegant approach to storing solar energy. The most efficient strategy is to couple the hydrogen-producing enzyme, the hydrogenase (H2ase), directly to photosystem I (PSI), which is a light-driven nanomachine found in photosynthetic organisms. PSI–H2ase fusions have been tested in vivo and in vitro. Both approaches have each their specific advantages and drawbacks. Here, a system to combine both approaches by assembling PSI–H2ase fusions in vivo for in vitro photoH2 production is established. For this, cyanobacterial PSI–H2ase fusion mutants are generated and characterized concerning photoH2 production in vivo. The chimeric protein is purified and embedded in a redox polymer on an electrode where it successfully produces photoH2 in vitro. The combination of in vivo and in vitro processes comes along with reciprocal benefits. The in vivo assembly ensures that the chimeric protein is fully functional and suited for the fabrication of bioelectrodes in vitro. At the same time, the photoelectrochemical in vitro characterization now permits to analyze the assemblies in detail. This will open avenues to optimize in vivo and in vitro approaches for photoH2 production in a target-oriented manner in the future.publishersversionpublishe

Synechocystis sp. PCC 6803 Requires the Bidirectional Hydrogenase to Metabolize Glucose and Arginine Under Oxic Conditions

The cyanobacterium Synechocystis sp.PCC 6803 possesses a bidirectional NiFe-hydrogenase, HoxEFUYH. It functions to produce hydrogen under dark, fermentative conditions and photoproduces hydrogen when dark-adapted cells are illuminated. Unexpectedly, we found that the deletion of the large subunit of the hydrogenase (HoxH) in Synechocystis leads to an inability to grow on arginine and glucose under continuous light in the presence of oxygen. This is surprising, as the hydrogenase is an oxygen-sensitive enzyme. In wild-type (WT) cells, thylakoid membranes largely disappeared, cyanophycin accumulated, and the plastoquinone (PQ) pool was highly reduced, whereas ΔhoxH cells entered a dormant-like state and neither consumed glucose nor arginine at comparable rates to the WT. Hydrogen production was not traceable in the WT under these conditions. We tested and could show that the hydrogenase does not work as an oxidase on arginine and glucose but has an impact on the redox states of photosynthetic complexes in the presence of oxygen. It acts as an electron valve as an immediate response to the supply of arginine and glucose but supports the input of electrons from arginine and glucose oxidation into the photosynthetic electron chain in the long run, possibly via the NDH-1 complex. Despite the data presented in this study, the latter scenario requires further proof. The exact role of the hydrogenase in the presence of arginine and glucose remains unresolved. In addition, a unique feature of the hydrogenase is its ability to shift electrons between NAD(H), NADP(H), ferredoxin, and flavodoxin, which was recently shown in vitro and might be required for fine-tuning. Taken together, our data show that Synechocystis depends on the hydrogenase to metabolize organic carbon and nitrogen in the presence of oxygen, which might be an explanation for its prevalence in aerobic cyanobacteria

Pyruvate: ferredoxin oxidoreductase and low abundant ferredoxins support aerobic photomixotrophic growth in cyanobacteria

The decarboxylation of pyruvate is a central reaction in the carbon metabolism of all organisms. It is catalyzed by the pyruvate:ferredoxin oxidoreductase (PFOR) and the pyruvate dehydrogenase (PDH) complex. Whereas PFOR reduces ferredoxin, the PDH complex utilizes NAD+. Anaerobes rely on PFOR, which was replaced during evolution by the PDH complex found in aerobes. Cyanobacteria possess both enzyme systems. Our data challenge the view that PFOR is exclusively utilized for fermentation. Instead, we show, that the cyanobacterial PFOR is stable in the presence of oxygen in vitro and is required for optimal photomixotrophic growth under aerobic and highly reducing conditions while the PDH complex is inactivated. We found that cells rely on a general shift from utilizing NAD(H)- to ferredoxin-dependent enzymes under these conditions. The utilization of ferredoxins instead of NAD(H) saves a greater share of the Gibbs-free energy, instead of wasting it as heat. This obviously simultaneously decelerates metabolic reactions as they operate closer to their thermodynamic equilibrium. It is common thought that during evolution, ferredoxins were replaced by NAD(P)H due to their higher stability in an oxidizing atmosphere. However, the utilization of NAD(P)H could also have been favored due to a higher competitiveness because of an accelerated metabolism.</p

Probing the role of the band 7 protein superfamily in the Cyanobacterium Synechocystis

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Investigating the Early Stages of Photosystem II Assembly in Synechocystis sp. PCC 6803: ISOLATION OF CP47 AND CP43 COMPLEXES*

Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b559, with CP47 binding first. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and β-carotene. A comparison of the low temperature absorption and fluorescence spectra in the QY region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1–3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex