Catalytic intermediates of cytochrome bd terminal oxidase at steady-state

The cytochrome bd ubiquinol oxidase from Escherichia coli couples the exergonic reduction of O2 to 2H2O to proton motive force generation by transmembrane charge separation. The oxidase contains hemes b558 and b595 and heme d, where O2 chemistry occurs through sequential formation of a few catalytic intermediates. The steady-state behavior of the isolated cytochrome bd has been examined by stopped-flow multiwavelength absorption spectroscopy. Under turnover conditions sustained by O2 and dithiothreitol-reduced ubiquinone, we found [1] that the mostly populated catalytic intermediates are the ferryl and oxy-ferrous species, with a minor fraction of the enzyme containing ferric heme d and possibly one electron on heme b558. These new findings clearly differ from those obtained with mammalian cytochrome c oxidase [2], in which the oxygen intermediates were not found to be populated at detectable levels under similar conditions. The results are discussed in the light of previously proposed models of the cytochrome bd catalytic cycle.