« lessĬyanobacterial Hox is a hydrogenase that consists of the hydrogen (H 2)-activating subunits HoxYH, which form a complex with the Ho圎FU assembly to mediate reactions with soluble electron carriers like NAD(P)H and ferredoxin (Fdx), thereby coupling photosynthetic electron transfer to energy-transforming catalytic reactions. 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. 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. The exact role of the hydrogenase in the presence of arginine and glucose remains unresolved. Despite the data presented in this study, the latter scenario requires further proof. 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. 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. Hydrogen production was not traceable in the WT under these conditions. 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 more » nor arginine at comparable rates to the WT. This is surprising, as the hydrogenase is an oxygen-sensitive enzyme. 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. It functions to produce hydrogen under dark, fermentative conditions and photoproduces hydrogen when dark-adapted cells are illuminated. The cyanobacterium Synechocystis sp.PCC 6803 possesses a bidirectional NiFe-hydrogenase, Ho圎FUYH. Altogether, the results provide new insights on the properties of Flv1 and Flv3 that enable tight control of reactivity for the complete reduction of oxygen to water, and in this capacity more » help preserve photosynthetic electron transport function. Titrations with NAD(P)H resulted in reduction of the diiron site without the accumulation of stable, reduced flavin intermediates. Based on mass spectrometry generated structural models, each Flv assembles as a homodimer with two oxidoreductase domains capable of binding two molecules of NAD(P)H per subunit, and the flavins are arranged to support electron transfer to the diiron sites for oxygen reduction. Reaction velocity curves were sigmoidal and Flv binding of NAD(P)H was cooperative. Under an oxygen atmosphere, Flv1 and Flv3 were found to catalyze ORR with either NADH or NADPH as the electron donor. To determine how these functions may be related, we investigated the kinetic properties of Flv1 and Flv3 from Synechocystis sp. The activity of Flvs have also been observed to form an important catalytic redox loop with water oxidation necessary for preserving photosynthetic electron transport function in cyanobacteria. Photosynthetic flavodiiron (Flv) proteins bind flavin and non-heme Fe cofactors and catalyze the oxygen reduction reaction (ORR) coupled to oxidation of reduced pyridine nucleotides during photosynthetic growth.
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