Cytochrome c oxidase from Rhodobacter sphaeroides is frequently used to model the more complex mitochondrial enzyme. The O2 reduction in both enzymes is generally described by a unidirectional mechanism involving the sequential formation of the ferrous-oxy complex (compound A), the PR state, the oxyferryl F form, and the oxidized state. In this study we investigated the reaction of dioxygen with the wild-type reduced R. sphaeroides cytochrome oxidase and the EQ(I-286) mutant using the CO flow-flash technique. Singular value decomposition and multiexponential fitting of the time-resolved optical absorption difference spectra showed that three apparent lifetimes, 18 μs, 53 μs, and 1.3 ms, are sufficient to fit the kinetics of the O 2 reaction of the wild-type enzyme. A comparison of the experimental intermediate spectra with the corresponding intermediate spectra of the bovine enzyme revealed that PR is not present in the reaction mechanism of the wild-type R. sphaeroides aa3. Transient absorbance changes at 440 and 610 nm support this conclusion. For the EQ(I-286) mutant, in which a key glutamic residue in the D proton pathway is replaced by glutamine, two lifetimes, 16 and 108 μs, were observed. A spectral analysis of the intermediates shows that the O2 reaction in the EQ(I-286) mutant terminates at the PR state, with 70% of heme a becoming oxidized. These results indicate significant differences in the kinetics of O2 reduction between the bovine and wild-type R. sphaeroides aa3 oxidases, which may arise from differences in the relative rates of internal electron and proton movements in the two enzymes.
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