Spectra of intermediates in oxidation and reduction of cytochrome c oxidase.
Two kinetic components with distinct difference spectra occur during reduction of cytochrome c oxidase by ruthenium hexamine. They are attributed to reduction of heme a (fast phase) and heme a3 (slow phase) (Scott, R. A., and Gray, H. B. (1980) J. Am. Chem. Soc. 102, 3219-3774). Two spectra seen during oxidation of cytochrome c oxidase by molecular oxygen have also been attributed to oxidation of hemes a3 and a (Greenwood, C., and Gibson, Q. H. (1967) J. Biol. Chem. 242, 1782-1787). We now report that spectra for the reductive and oxidative reactions obtained with the same preparations and the same apparatus under similar conditions are significantly different. The reactions appear to populate different reaction intermediates. Reconstitution into phospholipid vesicles does not affect these two spectra significantly. During turnover, the chief intermediates are those of the reductive pathway (Scott and Gray type intermediates). Reduction of heme a3 occurs approximately 70 times faster after turnover than the reduction of the resting enzyme. This is probably a dramatic "pulsing" effect (Wilson, M. T., Peterson, J., Antonini, E., Brunori, M., Colosimo, A., and Wyman, J. (1981) Proc. Natl. Acad. Sci. U.S.A. 7115-7118).