title: Formation of a cytochrome c-nitrous oxide reductase complex is obligatory for N2O reduction by Paracoccus pantotrophus.
abstract: Nitrous oxide reductase (N2OR) catalyses the final step of bacterial denitrification, the two-electron reduction of nitrous oxide (N2O) to dinitrogen (N2). N2OR contains two metal centers; a binuclear copper center, CuA, that serves to receive electrons from soluble donors, and a tetranuclear copper-sulfide center, CuZ, at the active site. Stopped flow experiments at low ionic strengths reveal rapid electron transfer (kobs=150 s-1) between reduced horse heart (HH) cytochrome c and the CuA center in fully oxidized N2OR. When fully reduced N2OR was mixed with oxidized cytochrome c, a similar rate of electron transfer was recorded for the reverse reaction, followed by a much slower internal electron transfer from CuZ to CuA(kobs=0.1-0.4 s-1). The internal electron transfer process is likely to represent the rate-determining step in the catalytic cycle. Remarkably, in the absence of cytochrome c, fully reduced N2OR is inert towards its substrate, even though sufficient electrons are stored to initiate a single turnover. However, in the presence of reduced cytochrome c and N2O, a single turnover occurs after a lag-phase. We propose that a conformational change in N2OR is induced by its specific interaction with cytochrome c that in turn either permits electron transfer between CuA and CuZ or controls the rate of N2O decomposition at the active site.
authors.affiliation: Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich, UK NR4 7TJ.
authors.forename: Tim
authors.initials: T
authors.lastname: Rasmussen
authors.forename: Thomas
authors.initials: T
authors.lastname: Brittain
authors.forename: Ben C
authors.initials: BC
authors.lastname: Berks
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Watmough
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thomson
mesh_terms: D000818:Animals; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism*; D004583:Electrons; D006736:Horses; D008958:Models, Molecular; D009609:Nitrous Oxide / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D042084:Paracoccus pantotrophus / Q000201:enzymology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D045304:Cytochromes c; D010088:Oxidoreductases; C022763:nitrous oxide reductase; D009609:Nitrous Oxide