title: Major Mo(V) EPR signature of Rhodobacter sphaeroides periplasmic nitrate reductase arising from a dead-end species that activates upon reduction. Relation to other molybdoenzymes from the DMSO reductase family.
abstract: Enzymes of the DMSO reductase family use a mononuclear Mo-bis(molybdopterin) cofactor (MoCo) to catalyze a variety of oxo-transfer reactions. Much functional information on nitrate reductase, one of the most studied members of this family, has been gained from EPR spectroscopy, but this technique is not always conclusive because the signature of the MoCo is heterogeneous, and which signals correspond to active species is still unsure. We used site-directed mutagenesis, EPR and protein film voltammetry to demonstrate that the MoCo in R. sphaeroides periplasmic nitrate reductase (NapAB) is subject to an irreversible reductive activation process whose kinetics we precisely define. This activation quantitatively correlates with the disappearance of the so-called "Mo(V) high-g" EPR signal, but this reductive process is too slow to be part of the normal catalytic cycle. Therefore, in NapAB, this most intense and most commonly observed signature of the MoCo arises from a dead-end, inactive state that gives a catalytically competent species only after reduction. This activation proceeds, even without substrate, according to a reduction followed by an irreversible nonredox step, both of which are pH independent. An apparently similar process occurs in other nitrate reductases (both assimilatory and membrane bound), and this also recalls the redox cycling procedure, which activates periplasmic DMSO reductases and simplifies their spectroscopic signatures. Hence we propose that heterogeneity at the active site and reductive activation are common properties of enzymes from the DMSO reductase family. Regarding NapAB, the fact that we could detect no Mo EPR signal upon reoxidizing the fully reduced enzyme suggests that the catalytically active form of the Mo(V) is thermodynamically unstable, as is the case for other enzymes of the DMSO reductase family. Our original approach, which combines spectroscopy and protein film voltammetry, proves useful for discriminating the forms of the active site on the basis of their catalytic properties. This could be applied to other enzymes for which the question arises as to the catalytic relevance of certain long-lived, spectroscopically characterized species.
authors.affiliation: Unité de Bioénergétique et Ingénierie des Protéines, IBSM, UPR 9036, CNRS, 31 Chemin Joseph Aiguier, F-13402 Marseille Cedex 20, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Fourmond
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Burlat
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dementin
authors.forename: Pascal
authors.initials: P
authors.lastname: Arnoux
authors.forename: Monique
authors.initials: M
authors.lastname: Sabaty
authors.forename: Séverine
authors.initials: S
authors.lastname: Boiry
authors.forename: Bruno
authors.initials: B
authors.lastname: Guigliarelli
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: D000327:Adsorption; D000465:Algorithms; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D008872:Microwaves; D008982:Molybdenum / Q000737:chemistry*; D009154:Mutation; D050901:Nitrate Reductase / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D011199:Potentiometry; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics
chemical_list: D007506:Iron-Sulfur Proteins; D008982:Molybdenum; D010088:Oxidoreductases; D050901:Nitrate Reductase; C045664:dimethyl sulfoxide reductase