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The cyanobacterium Synechocystis sp. PCC 6803 has a bidirectional [NiFe]-hydrogenase (Hox hydrogenase) which reversibly reduces protons to H2. This enzyme is composed of a hydrogenase domain and a diaphorase moiety, which is distinctly homologous to the NADH input module of mitochondrial respiratory Complex I. Hox hydrogenase physiological function is still unclear, since it is not required for Synechocystis fitness under standard growth conditions. We analyzed the phenotype under prolonged darkness of three Synechocystis knock-out strains, lacking either Hox hydrogenase (ΔHoxE-H) or one of the proteins responsible for the assembly of its NiFe active site (ΔHypA1 and ΔHypB1). We found that Hox hydrogenase is required for Synechocystis growth under this condition, regardless of the functional status of its catalytic site, suggesting an additional role beside hydrogen metabolism. Moreover, quantitative proteomic analyses revealed that the expression levels of several subunits of the respiratory NADPH/plastoquinone oxidoreductase (NDH-1) are reduced when Synechocystis is grown in the dark. Our findings suggest that the Hox hydrogenase could contribute to electron transport regulation when both photosynthetic and respiratory pathways are down-regulated, and provide a possible explanation for the close evolutionary relationship between mitochondrial respiratory Complex I and cyanobacterial [NiFe]-hydrogenases.

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[FeFe]-hydrogenases catalyse the reduction of protons to hydrogen at a complex 2Fe[4Fe4S] center called H-cluster. The assembly of this active site is a multistep process involving three proteins, HydE, HydF and HydG. According to the current models, HydF has the key double role of scaffold, upon which the final H-cluster precursor is assembled, and carrier to transfer it to the target hydrogenase. The X-ray structure of HydF indicates that the protein is a homodimer with both monomers carrying two functional domains: a C-terminal FeS cluster-binding domain, where the precursor is assembled, and a N-terminal GTPase domain, whose exact contribution to cluster biogenesis and hydrogenase activation is still elusive. We previously obtained several hints suggesting that the binding of GTP to HydF could be involved in the interactions of this scaffold protein with the other maturases and with the hydrogenase itself. In this work, by means of site directed spin labeling coupled to EPR/PELDOR spectroscopy, we explored the conformational changes induced in a recombinant HydF protein by GTP binding, and provide the first clue that the HydF GTPase domain could be involved in the H-cluster assembly working as a molecular switch similarly to other known small GTPases.Biohydrogen production, one of the most promising frontiers in the field of renewable energies, is achieved in nature by several prokaryotic and eukaryotic microorganisms through a general class of evolutionarily unrelated metalloenzymes called hydrogenases. Among them, [FeFe]-and [NiFe]-hydrogenases are the most widespread 1 . These proteins have different distribution, catalytic properties and molecular architectures, but they are both able to reversibly reduce protons to H 2 by means of metal clusters with some key similarities, including the coordination to the polypeptide chain through four conserved cysteine residues and the presence of iron-bound CO and CN − ligands 2 . In the case of [FeFe]-hydrogenases, the active site (referred to as the H-cluster) is particularly complex since it is composed of a [4Fe4S] cubane linked via a cysteine bridge to a 2Fe subcluster comprising the CO and CN -molecules and an additional dithiomethylamine ligand 3-5 . This 2Fe subcluster needs a specific set of maturation proteins to be first assembled in the active form and then inserted into the target functional [FeFe]-hydrogenase. Three conserved proteins are involved in its biosynthesis and delivery, i.e. HydE, HydF and HydG, discovered in the unicellular green alga Chlamydomonas reinhardtii and then found in all microorganisms containing a [FeFe]-hydrogenase 6 . Both HydE and HydG are radical S-adenosylmethionine (SAM) proteins whereas HydF is a GTPase carrying a [4Fe4S] cluster binding motif 6 . The 3D crystal structures of all these maturases have been solved [7][8][9][10] , and several in vitro and cell-free experiments using purified recombinant proteins have been performed to understand their functions, allowing to propose a two-steps model that describes a H-cluste...