Catalytic intermediates of cytochrome bd terminal oxidase at steady-state: Ferryl and oxy-ferrous species dominate

• Published in: Biochimica et biophysica acta 2011-05, Vol.1807 (5), p.503-509
• Main Authors: Borisov, Vitaliy B., Forte, Elena, Sarti, Paolo, Giuffrè, Alessandro
• Format: Article
• Language: English
• Subjects: absorption; Biocatalysis; Catalytic turnover; Chlorin; cytochrome-c oxidase; Cytochromes - metabolism; dithiothreitol; Electron Transport Chain Complex Proteins - metabolism; Electron Transport Complex IV - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli Proteins - metabolism; Ferrous Compounds - metabolism; heme; Hemoprotein; mammals; oxidation; Oxidoreductases - metabolism; oxygen; Oxygen chemistry; proton-motive force; Reaction mechanism; Respiration; spectroscopy; ubiquinones
• ISSN: 0005-2728; 0006-3002; 1879-2650
• DOI: 10.1016/j.bbabio.2011.02.007

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 2O 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. ► Spectral features of cytochrome bd terminal oxidase examined at steady-state by time-resolved spectrophotometry ► Ferryl and oxy-ferrous catalytic intermediates dominate ► Model of the catalytic cycle proposed.