title: Reassessing the strategies for trapping catalytic intermediates during nitrate reductase turnover.
abstract: We examined the kinetics of nitrate reduction by periplasmic nitrate reductase (Nap) by using protein film voltammetry and solution assays. We demonstrate that, under turnover conditions, the enzyme exists as a mixture of active and inactive forms which interconvert on a time scale that is much slower than turnover. The dead-end species accumulates under mildly reducing conditions and at high nitrate concentration, resulting in substrate inhibition and in an uncommon hysteresis in the voltammetric signature. Solution assays with two electron donors having different reduction potentials fully support the electrochemical results. This illustrates the consequences of the high flexibility of the active site molybdenum coordination sphere and questions the conclusions from earlier studies in which attempts were made to trap catalytic intermediates of Nap in experiments carried out under turnover conditions at very high substrate concentration.
authors.affiliation: Unité de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, Centre National de la Recherche Scientifique, UPR 9036, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Fourmond
authors.forename: Monique
authors.initials: M
authors.lastname: Sabaty
authors.forename: Pascal
authors.initials: P
authors.lastname: Arnoux
authors.forename: Patrick
authors.initials: P
authors.lastname: Bertrand
authors.forename: David
authors.initials: D
authors.lastname: Pignol
authors.forename: Christophe
authors.initials: C
authors.lastname: Léger
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D055664:Electrochemical Techniques; D007700:Kinetics; D008982:Molybdenum / Q000737:chemistry; D050901:Nitrate Reductase / Q000737:chemistry* / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D010084:Oxidation-Reduction; D019897:Periplasm / Q000201:enzymology
chemical_list: D009566:Nitrates; D008982:Molybdenum; D050901:Nitrate Reductase