| Cytochrome c oxidase (CcO) catalyzes the four-electron, four-proton reduction of dioxygen to water. We have studied single-turnover events during the reduction of O2 using two approaches: (1) A caged oxygen approach in which a dioxygen precursor is photolyzed to produce molecular oxygen within nanoseconds, and (2) a flow-flash approach in which carbon monoxide is photolyzed from heme a3 of CcO in the presence of O2.;In the caged oxygen approach, time-resolved optical absorption difference spectra were recorded following laser photolysis of a (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)cobalt(III)] complex in the presence of reduced CcO. The results were compared to conventional flow-flash experiments. Our results indicate that ∼90 muM O2 can be generated with a single laser pulse and that this approach is useful for studying both heme-copper oxidases and other fast biological reactions involving O2.;To investigate intermediates generated during dioxygen reduction by CcO, CO flow-flash experiments were performed on the two-electron- (M) and four-electron-reduced (R) enzyme. When the time-resolved optical absorption difference spectra are analyzed using a unidirectional sequential mechanism, the spectral properties of the "peroxy" intermediate (PM) were found to be similar to that of the bench-made "peroxy" (CcO-607) form, but distinct from the corresponding PR.;In order to investigate the nature of the PR intermediate in more detail, the reduction of dioxygen by four-electron-reduced CcO at three pHs, 6.2, 7.5, and 8.5 was carried out using the flow-flash technique. When the kinetics were analyzed using a unidirectional sequential model, the putative CcO-607 intermediate (PR) was best represented by a pH-dependent mixture of compound A (the O2-bound form) and the bench-made CcO-607 and CcO-580 species. The ferryl form was favored at low pH. To account for these observations, we propose a kinetic model that consists of two pathways, one producing CcO-607 and the other CcO-580, both from compound A. The two pathways in this model are interconnected and the rate of exchange between the two is pH-dependent. |
