title: Mechanism of inhibition of NiFe hydrogenase by nitric oxide.
abstract: Hydrogenases reversibly catalyze the oxidation of molecular hydrogen and are inhibited by several small molecules including O2, CO and NO. In the present work, we investigate the mechanism of inhibition by NO of the oxygen-sensitive NiFe hydrogenase from Desulfovibrio fructosovorans by coupling site-directed mutagenesis, protein film voltammetry (PFV) and EPR spectroscopy. We show that micromolar NO strongly inhibits NiFe hydrogenase and that the mechanism of inhibition is complex, with NO targeting several metallic sites in the protein. NO reacts readily at the NiFe active site according to a two-step mechanism. The first and faster step is the reversible binding of NO to the active site followed by a slower and irreversible transformation at the active site. NO also induces irreversible damage of the iron-sulfur centers chain. We give direct evidence of preferential nitrosylation of the medial [3Fe-4S] to form dinitrosyl-iron complexes.
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 Chemin J. Aiguier, F-13402 Marseille Cedex 20, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Ceccaldi
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 Chemin J. Aiguier, F-13402 Marseille Cedex 20, France.
authors.forename: Emilien
authors.initials: E
authors.lastname: Etienne
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 Chemin J. Aiguier, F-13402 Marseille Cedex 20, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dementin
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 Chemin J. Aiguier, F-13402 Marseille Cedex 20, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Guigliarelli
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 Chemin J. Aiguier, F-13402 Marseille Cedex 20, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Léger
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 Chemin J. Aiguier, F-13402 Marseille Cedex 20, France. Electronic address: burlat@imm.cnrs.fr.
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Burlat
mesh_terms: D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D009569:Nitric Oxide / Q000494:pharmacology*
chemical_list: D009569:Nitric Oxide; C043296:nickel-iron hydrogenase; D006864:Hydrogenase
keywords: EPR spectroscopy; Hydrogenase; Inhibition mechanism; Metalloenzyme; Nitric oxide; Protein film voltammetry