title: Reductively activated nitrous oxide reductase reacts directly with substrate.
abstract: In the terminal step of bacterial denitrification, N2O is converted to N2 at the mu4-sulfide bridged tetranuclear CuZ center of nitrous oxide reductase. The enzyme can be activated by reduced methyl viologen, with up to a 15-fold increase in specific activity. The reductively activated nitrous oxide reductase from Achromobacter cycloclastes was isolated and characterized by visible absorption and EPR spectroscopy, and both methods showed that the CuZ center can attain a [4Cu(I)] oxidation state. When N2O was added to the activated, reductant-free enzyme, distinct spectral changes were observed, indicating that this state of the enzyme interacts with substrate. This was further supported by the detection of 15N-labeled product in the absence of steady-state turnover conditions. A new absorption band around 970 nm appeared following reaction of activated nitrous oxide reductase with N2O, which may represent a catalytic intermediate state of the enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, USA.
authors.forename: Jeannine M
authors.initials: JM
authors.lastname: Chan
authors.forename: John A
authors.initials: JA
authors.lastname: Bollinger
authors.forename: Cassidy L
authors.initials: CL
authors.lastname: Grewell
authors.forename: David M
authors.initials: DM
authors.lastname: Dooley
mesh_terms: D042422:Achromobacter cycloclastes / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D007700:Kinetics; D009584:Nitrogen / Q000737:chemistry / Q000378:metabolism; D009609:Nitrous Oxide / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D013053:Spectrophotometry; D013379:Substrate Specificity
chemical_list: D010088:Oxidoreductases; C022763:nitrous oxide reductase; D009609:Nitrous Oxide; D009584:Nitrogen