title: Reductive activation of E. coli respiratory nitrate reductase.
abstract: Over the past decades, a number of authors have reported the presence of inactive species in as-prepared samples of members of the Mo/W-bisPGD enzyme family. This greatly complicated the spectroscopic studies of these enzymes, since it is impossible to discriminate between active and inactive species on the basis of the spectroscopic signatures alone. Escherichia coli nitrate reductase A (NarGHI) is a member of the Mo/W-bisPGD family that allows anaerobic respiration using nitrate as terminal electron acceptor. Here, using protein film voltammetry on NarGH films, we show that the enzyme is purified in a functionally heterogeneous form that contains between 20 and 40% of inactive species that activate the first time they are reduced. This activation proceeds in two steps: a non-redox reversible reaction followed by an irreversible reduction. By carefully correlating electrochemical and EPR spectroscopic data, we show that neither the two major Mo(V) signals nor those of the two FeS clusters that are the closest to the Mo center are associated with the two inactive species. We also conclusively exclude the possibility that the major "low-pH" and "high-pH" Mo(V) EPR signatures correspond to species in acid-base equilibrium.
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 chemin J. Aiguier, F-13402 Marseille cedex 20, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Ceccaldi
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 chemin J. Aiguier, F-13402 Marseille cedex 20, France.
authors.forename: Julia
authors.initials: J
authors.lastname: Rendon
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, LCB UMR 7283, 31 chemin J. Aiguier, F-13402 Marseille cedex 20, France.
authors.forename: René
authors.initials: R
authors.lastname: Toci
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, LCB UMR 7283, 31 chemin J. Aiguier, F-13402 Marseille cedex 20, France.
authors.forename: Axel
authors.initials: A
authors.lastname: Magalon
authors.affiliation: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 chemin J. Aiguier, F-13402 Marseille cedex 20, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Grimaldi
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
keywords: EPR spectroscopy; Mo/W bisPGD enzyme family; Protein film voltammetry; Respiratory nitrate reductase