title: A threonine stabilizes the NiC and NiR catalytic intermediates of [NiFe]-hydrogenase.
abstract: The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production.
authors: From the Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, UMR 7281 Aix-Marseille Université/CNRS, 31 Chemin J. Aiguier, 13402 Marseille Cedex 20, France.
authors: Abbas
authors: A
authors: Abou-Hamdan
authors: From the Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, UMR 7281 Aix-Marseille Université/CNRS, 31 Chemin J. Aiguier, 13402 Marseille Cedex 20, France.
authors: Pierre
authors: P
authors: Ceccaldi
authors: Metalloproteins Unit, Institut de Biologie Structurale, UMR 5075 Commissariat à l'Energie Atomique/CNRS/Université Joseph Fourier, 6 rue Jules Horowitz, 38000, Grenoble, France.
authors: Hugo
authors: H
authors: Lebrette
authors: Instituto de Catálisis y Petroleoquímica, Consejo Superior de Investigaciones Científicas, c/Marie Curie 2, L10, 28049 Madrid, Spain.
authors: Oscar
authors: O
authors: Gutiérrez-Sanz
authors: Laboratoire de Bioénergétique et Biotechnologie des Bactéries et Microalgues, Commissariat à l'Energie Atomique, Direction des Sciences du Vivant, Institut de Biologie Environnementale et Biotechnologie, Saint-Paul-lez-Durance, F-13108, France CNRS, UMR 7265 Biologie Végétale et Microbiologie Environnementale (BVME), Saint-Paul-lez-Durance, 13108, France, and Aix-Marseille Université, BVME UMR7265, Marseille F-13284, France.
authors: Pierre
authors: P
authors: Richaud
authors: Laboratoire de Bioénergétique et Biotechnologie des Bactéries et Microalgues, Commissariat à l'Energie Atomique, Direction des Sciences du Vivant, Institut de Biologie Environnementale et Biotechnologie, Saint-Paul-lez-Durance, F-13108, France CNRS, UMR 7265 Biologie Végétale et Microbiologie Environnementale (BVME), Saint-Paul-lez-Durance, 13108, France, and Aix-Marseille Université, BVME UMR7265, Marseille F-13284, France.
authors: Laurent
authors: L
authors: Cournac
authors: From the Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, UMR 7281 Aix-Marseille Université/CNRS, 31 Chemin J. Aiguier, 13402 Marseille Cedex 20, France.
authors: Bruno
authors: B
authors: Guigliarelli
authors: Instituto de Catálisis y Petroleoquímica, Consejo Superior de Investigaciones Científicas, c/Marie Curie 2, L10, 28049 Madrid, Spain.
authors: Antonio L
authors: AL
authors: De Lacey
authors: From the Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, UMR 7281 Aix-Marseille Université/CNRS, 31 Chemin J. Aiguier, 13402 Marseille Cedex 20, France.
authors: Christophe
authors: C
authors: Léger
authors: Metalloproteins Unit, Institut de Biologie Structurale, UMR 5075 Commissariat à l'Energie Atomique/CNRS/Université Joseph Fourier, 6 rue Jules Horowitz, 38000, Grenoble, France.
authors: Anne
authors: A
authors: Volbeda
authors: From the Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, UMR 7281 Aix-Marseille Université/CNRS, 31 Chemin J. Aiguier, 13402 Marseille Cedex 20, France.
authors: Bénédicte
authors: B
authors: Burlat
authors: From the Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, UMR 7281 Aix-Marseille Université/CNRS, 31 Chemin J. Aiguier, 13402 Marseille Cedex 20, France, dementin@imm.cnrs.fr.
authors: Sébastien
authors: S
authors: Dementin
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry*; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003901:Desulfovibrio / Q000201:enzymology*; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D006864:Hydrogenase / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D013912:Threonine / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D013912:Threonine; C043296:nickel-iron hydrogenase; D006864:Hydrogenase
keywords: Electron Paramagnetic Resonance (EPR); Enzyme Kinetics; Fourier Transform IR (FTIR); Hydrogenase; X-ray Crystallography