title: Reductive activation in periplasmic nitrate reductase involves chemical modifications of the Mo-cofactor beyond the first coordination sphere of the metal ion.
abstract: In Rhodobacter sphaeroides periplasmic nitrate reductase NapAB, the major Mo(V) form (the "high g" species) in air-purified samples is inactive and requires reduction to irreversibly convert into a catalytically competent form (Fourmond et al., J. Phys. Chem., 2008). In the present work, we study the kinetics of the activation process by combining EPR spectroscopy and direct electrochemistry. Upon reduction, the Mo (V) "high g" resting EPR signal slowly decays while the other redox centers of the protein are rapidly reduced, which we interpret as a slow and gated (or coupled) intramolecular electron transfer between the [4Fe-4S] center and the Mo cofactor in the inactive enzyme. Besides, we detect spin-spin interactions between the Mo(V) ion and the [4Fe-4S](1+) cluster which are modified upon activation of the enzyme, while the EPR signatures associated to the Mo cofactor remain almost unchanged. This shows that the activation process, which modifies the exchange coupling pathway between the Mo and the [4Fe-4S](1+) centers, occurs further away than in the first coordination sphere of the Mo ion. Relying on structural data and studies on Mo-pyranopterin and models, we propose a molecular mechanism of activation which involves the pyranopterin moiety of the molybdenum cofactor that is proximal to the [4Fe-4S] cluster. The mechanism implies both the cyclization of the pyran ring and the reduction of the oxidized pterin to give the competent tricyclic tetrahydropyranopterin form.
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 chemin J. Aiguier, F-13402 Marseille cedex 20, France.
authors.forename: Julien G J
authors.initials: JG
authors.lastname: Jacques
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 chemin J. Aiguier, F-13402 Marseille cedex 20, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Fourmond
authors.affiliation: Aix-Marseille Université, CNRS, CEA, DSV/IBEB/LBC UMR 7265, F-13108 Saint Paul Lez Durance, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Arnoux
authors.affiliation: Aix-Marseille Université, CNRS, CEA, DSV/IBEB/LBC UMR 7265, F-13108 Saint Paul Lez Durance, France.
authors.forename: Monique
authors.initials: M
authors.lastname: Sabaty
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, CEA, DSV/IBEB/LBC UMR 7265, F-13108 Saint Paul Lez Durance, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Grosse
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 chemin J. Aiguier, F-13402 Marseille cedex 20, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Biaso
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 chemin J. Aiguier, F-13402 Marseille cedex 20, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Bertrand
authors.affiliation: Aix-Marseille Université, CNRS, CEA, DSV/IBEB/LBC UMR 7265, F-13108 Saint Paul Lez Durance, France.
authors.forename: David
authors.initials: D
authors.lastname: Pignol
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.
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. Electronic address: burlat@imm.cnrs.fr.
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Burlat
mesh_terms: D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism*; D055664:Electrochemical Techniques; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D007477:Ions; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008982:Molybdenum / Q000378:metabolism*; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D019897:Periplasm / Q000201:enzymology*; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism*; D011622:Pterins / Q000737:chemistry / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013113:Spin Labels; D013696:Temperature
chemical_list: D003067:Coenzymes; D007477:Ions; D007506:Iron-Sulfur Proteins; D008024:Ligands; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase; D011621:Pteridines; D011622:Pterins; D013113:Spin Labels; C490076:pyranopterin; C022261:molybdopterin
keywords: EPR spectroscopy; Electron transfer; Molybdenum; Nitrate reductase; Protein film voltammetry; Pyranopterin