Multichannel Analysis of Single-Turnover Kinetics of Cytochrome aa_3 Reduction of O_2

The single-turnover kinetics of the oxidation of cytochrome aa_3 by O_2 have been studied using a new approach. Up to 1000 whole spectra covering both the Soret and α regions were sequentially collected at room temperature from single samples with a time resolution of 10 μs. All of the spectral and time information were used in analyses based on singular value decomposition. Four spectral transitions (i.e., intermediates) were distinguished with time constants near 0.01, 0.1, 1.1, and 30 ms. Two different kinds of sequential models were evaluated, one linear and the other branched. Although past kinetic analyses have emphasized the linear sequential model, the complexity of the intramolecular electron transfer in this enzyme suggests that a branched model be considered. This is especially true in a single-turnover experiment where earlier optical and EPR studies have pointed unequivocally to a branched model [Clore et al. (1980) Biochem. J. 185, 139−154; Blair et al. (1985) J. Am. Chem. Soc. 107, 7389−7399]. In the present study, analysis of spectral data in terms of the linear model did not reveal the formation and decay of the expected oxyferryl intermediate, whereas analysis of the branched model did. The results obtained using the branched model are consistent with all of the available evidence from a broad range of physical techniques that have been applied to examine the single-turnover kinetics of the oxidation of reduced cytochrome aa_3 by O_2.