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 a(3)(2+)-CO Cu-B(+) cytochrome a(3+) Cu-A(2+)) 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 a least two processes, with apparent lifetimes of 1.4 mu s 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 a(3) 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 mu s, 5.2 mu s, 83.7 mu s, 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 a(3), followed by electron transfer from cytochrome a(3) to cytochrome a with subsequent electron transfer to Cu-A. 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.