title: The H-bond network surrounding the pyranopterins modulates redox cooperativity in the molybdenum-bisPGD cofactor in arsenite oxidase.
abstract: While the molybdenum cofactor in the majority of bisPGD enzymes goes through two consecutive 1-electron redox transitions, previous protein-film voltammetric results indicated the possibility of cooperative (n=2) redox behavior in the bioenergetic enzyme arsenite oxidase (Aio). Combining equilibrium redox titrations, optical and EPR spectroscopies on concentrated samples obtained via heterologous expression, we unambiguously confirm this claim and quantify Aio's redox cooperativity. The stability constant, Ks, of the Mo(V) semi-reduced intermediate is found to be lower than 10(-3). Site-directed mutagenesis of residues in the vicinity of the Mo-cofactor demonstrates that the degree of redox cooperativity is sensitive to H-bonding interactions between the pyranopterin moieties and amino acid residues. Remarkably, in particular replacing the Gln-726 residue by Gly results in stabilization of (low-temperature) EPR-observable Mo(V) with KS=4. As evidenced by comparison of room temperature optical and low temperature EPR titrations, the degree of stabilization is temperature-dependent. This highlights the importance of room-temperature redox characterizations for correctly interpreting catalytic properties in this group of enzymes. Geochemical and phylogenetic data strongly indicate that molybdenum played an essential biocatalytic roles in early life. Molybdenum's redox versatility and in particular the ability to show cooperative (n=2) redox behavior provide a rationale for its paramount catalytic importance throughout the evolutionary history of life. Implications of the H-bonding network modulating Molybdenum's redox properties on details of a putative inorganic metabolism at life's origin are discussed.
authors.affiliation: CNRS, Aix-Marseille Univ, BIP UMR 7281, IMM FR 3479, 31 chemin J. Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Simon
authors.initials: S
authors.lastname: Duval
authors.affiliation: Institute of Structural and Molecular Biology, University College London, WC1E 6BT, UK.
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Santini
authors.affiliation: Institut de Biologie Environnementale et Biotechnologie, CEA de Cadarache, 13108 Saint Paul Lez Durance Cedex, France.
authors.forename: David
authors.initials: D
authors.lastname: Lemaire
authors.affiliation: IMBE Aix Marseille Université, IRD CNRS UAPV, Faculté de Pharmacie, 13005 Marseille, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Chaspoul
authors.affiliation: Jet Propulsion Laboratory, California Institute of Technology, 4800 Oak Grove Drive, Pasadena, CA 91109-8099, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Russell
authors.affiliation: CNRS, Aix-Marseille Univ, BIP UMR 7281, IMM FR 3479, 31 chemin J. Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Stephane
authors.initials: S
authors.lastname: Grimaldi
authors.affiliation: CNRS, Aix-Marseille Univ, BIP UMR 7281, IMM FR 3479, 31 chemin J. Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Nitschke
authors.affiliation: CNRS, Aix-Marseille Univ, BIP UMR 7281, IMM FR 3479, 31 chemin J. Aiguier, 13402 Marseille Cedex 20, France. Electronic address: schoepp@imm.cnrs.fr.
authors.forename: Barbara
authors.initials: B
authors.lastname: Schoepp-Cothenet
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D006860:Hydrogen Bonding; D008982:Molybdenum / Q000737:chemistry*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D011622:Pterins / Q000737:chemistry*
chemical_list: D011622:Pterins; C490076:pyranopterin; D008982:Molybdenum; D010088:Oxidoreductases; C078179:arsenite oxidase
keywords: Arsenite oxidase; EPR spectroscopy; Molybdenum enzyme; Optical spectroscopy; Redox titrations