title: The Carbon Monoxide Dehydrogenase from Desulfovibrio vulgaris.
abstract: Ni-containing Carbon Monoxide Dehydrogenases (CODHs) catalyze the reversible conversion between CO and CO₂and are involved in energy conservation and carbon fixation. These homodimeric enzymes house two NiFeS active sites (C-clusters) and three accessory [4Fe-4S] clusters. The Desulfovibrio vulgaris (Dv) genome contains a two-gene CODH operon coding for a CODH (cooS) and a maturation protein (cooC) involved in nickel insertion in the active site. According to the literature, the question of the precise function of CooC as a chaperone folding the C-cluster in a form which accommodates free nickel or as a mere nickel donor is not resolved. Here, we report the biochemical and spectroscopic characterization of two recombinant forms of the CODH, produced in the absence and in the presence of CooC, designated CooS and CooS(C), respectively. CooS contains no nickel and cannot be activated, supporting the idea that the role of CooC is to fold the C-cluster so that it can bind nickel. As expected, CooS(C) is Ni-loaded, reversibly converts CO and CO₂, displays the typical Cred1 and Cred2 EPR signatures of the C-cluster and activates in the presence of methyl viologen and CO in an autocatalytic process. However, Ni-loaded CooS(C) reaches maximum activity only upon reductive treatment in the presence of exogenous nickel, a phenomenon that had not been observed before. Surprisingly, the enzyme displays the Cred1 and Cred2 signatures whether it has been activated or not, showing that this activation process of the Ni-loaded Dv CODH is not associated with structural changes at the active site.
authors.affiliation: Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, UMR 7281, Aix-Marseille Université/CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Jessica
authors.initials: J
authors.lastname: Hadj-Saïd
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Maria-Eirini
authors.initials: ME
authors.lastname: Pandelia
authors.affiliation: Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, UMR 7281, Aix-Marseille Université/CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Léger
authors.affiliation: Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, UMR 7281, Aix-Marseille Université/CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Fourmond
authors.affiliation: Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, UMR 7281, Aix-Marseille Université/CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Electronic address: dementin@imm.cnrs.fr.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dementin
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D016969:Desulfovibrio vulgaris / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet
chemical_list: D009097:Multienzyme Complexes; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase
keywords: Electron paramagnetic resonance (EPR); Enzyme kinetics; Ni-containing Carbon Monoxide Dehydrogenase; Protein purification