Time-resolved optical absorption studies of intramolecular electron transfer in cytochrome c oxidase.

Intramolecular electron transfer and conformational changes in cytochrome c oxidase were studied at room temperature following the photodissociation of CO bound to mixed-valence enzyme (cytochrome a3(2+)-CO CuB+ cytochrome a3+ CuA2+) and fully reduced enzyme. Time-resolved optical absorption difference spectra were collected in the Soret region on time scales of nanoseconds to milliseconds using a gated optical spectrometric multichannel analyzer. A global exponential fitting procedure combined with a singular value decomposition method was used to analyze the transient difference spectra at various times following CO photolysis. The analysis shows that at least two processes, with apparent lifetimes of 1.4 microseconds and 11.1 ms, are present following the photodissociation of CO bound to the fully reduced enzyme. These are attributed to a conformational change and CO recombination at the cytochrome a3 site, respectively. Global analysis of the mixed-valence CO complex transient difference spectra showed the presence of five intermediates with apparent lifetimes of 1.0 microseconds, 5.2 microseconds, 83.7 microseconds, 10.5 ms, and 25.3 ms. The data on a microsecond time scale are consistent with a mechanism involving a conformational change at cytochrome a3, followed by electron transfer from cytochrome a3 to cytochrome a with subsequent electron transfer to CuA. One of the two processes on a millisecond time scale is attributed to CO recombination and the other to a structural rearrangement or heme-heme electron transfer. On the basis of this mechanism, the kinetics and the absorption spectra of the intermediates involved in the conformational and electron transfer dynamics of the mixed-valence enzyme were determined.