title: Kinetics of substrate inhibition of periplasmic nitrate reductase.
abstract: Periplasmic nitrate reductase catalyzes the reduction of nitrate into nitrite using a mononuclear molybdenum cofactor that has nearly the same structure in all enzymes of the DMSO reductase family. In previous electrochemical investigations, we found that the enzyme exists in several inactive states, some of which may have been previously isolated and mistaken for catalytic intermediates. In particular, the enzyme slowly and reversibly inactivates when exposed to high concentrations of nitrate. Here, we study the kinetics of substrate inhibition and its dependence on electrode potential and substrate concentration to learn about the properties of the active and inactive forms of the enzyme. We conclude that the substrate-inhibited enzyme never significantly accumulates in the EPR-active Mo(+V) state. This conclusion is relevant to spectroscopic investigations where attempts are made to trap a Mo(+V) catalytic intermediate using high concentrations of nitrate.
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: Bénédicte
authors.initials: B
authors.lastname: Burlat
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: 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, 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. Electronic address: vincent.fourmond@imm.cnrs.fr.
authors.forename: Vincent
authors.initials: V
authors.lastname: Fourmond
mesh_terms: D007700:Kinetics; D050901:Nitrate Reductase / Q000037:antagonists & inhibitors*; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction; D019897:Periplasm / Q000201:enzymology*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D009573:Nitrites; D050901:Nitrate Reductase
keywords: EPR spectroscopy; Electrochemistry; Enzyme kinetics; Nitrate reductase