title: Catalytic intermediates of cytochrome bd terminal oxidase at steady-state: ferryl and oxy-ferrous species dominate.
abstract: The cytochrome bd ubiquinol oxidase from Escherichia coli couples the exergonic two-electron oxidation of ubiquinol and four-electron reduction of O(2) to 2H(2)O to proton motive force generation by transmembrane charge separation. The oxidase contains two b-type hemes (b(558) and b(595)) and one heme d, where O(2) is captured and converted to water through sequential formation of a few intermediates. The spectral features of the isolated cytochrome bd at steady-state have been examined by stopped-flow multiwavelength absorption spectroscopy. Under turnover conditions, sustained by O(2) and dithiothreitol (DTT)-reduced ubiquinone, the ferryl and oxy-ferrous species are the mostly populated catalytic intermediates, with a residual minor fraction of the enzyme containing ferric heme d and possibly one electron on heme b(558). These findings are unprecedented and differ from those obtained with mammalian cytochrome c oxidase, in which the oxygen intermediates were not found to be populated at detectable levels under similar conditions [M.G. Mason, P. Nicholls, C.E. Cooper, The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres, Biochem. J. 422 (2009) 237-246]. The data on cytochrome bd are consistent with the observation that the purified enzyme has the heme d mainly in stable oxy-ferrous and ferryl states. The results are here discussed in the light of previously proposed models of the catalytic cycle of cytochrome bd.
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119991, Russian Federation.
authors.forename: Vitaliy B
authors.initials: VB
authors.lastname: Borisov
authors.forename: Elena
authors.initials: E
authors.lastname: Forte
authors.forename: Paolo
authors.initials: P
authors.lastname: Sarti
authors.forename: Alessandro
authors.initials: A
authors.lastname: Giuffrè
mesh_terms: D055162:Biocatalysis; D003573:Cytochrome b Group; D003580:Cytochromes / Q000378:metabolism*; D045222:Electron Transport Chain Complex Proteins / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005296:Ferrous Compounds / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D005296:Ferrous Compounds; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli; D003576:Electron Transport Complex IV