1. The reaction of the partially reduced mixed-valence state of cytochrome c oxidase (a3+CuA2+a3(2+)COCuB+) with O2 was studied by the rapid-reaction technique of flow-flash spectrophotometry at room temperature. Biphasic absorption records are observed in the time range up to 2 ms in both the Soret and visible spectral regions. The fast-phase rate is O2-concentration-dependent and reaches a pseudo-first-order value of 4.5 X 10(4)s-1 at 680 microM-O2 at 20 degrees C. Under the same conditions the second-phase rate is limited at 6.0 X 10(3)s-1. Kinetic difference spectra of the two species in the Soret region are not markedly different in form, whereas in the visible region two spectroscopically different species are clearly distinguished. 4. The first intermediate has a peak at 595 nm and a trough at 605 nm. The form of this spectrum resembles that seen in low-temperature studies and assigned to an O2-bound form of ferrocytochrome a3. This evidence supports a structure for oxycytochrome c oxidase with O2 bound only to cytochrome a3 and not bridged between cytochrome a3 and CuB. The second intermediate has a difference spectrum with a trough at 592 nm and a peak at 610 nm. Again, the form of this spectrum is similar to that observed during the O2 reaction at low temperature and is though to be a result of electron transfer from the oxidase to bound O2. 5. The oxygen profile of the fast phase suggests that a spectroscopically silent species may precede the formation of compound A. These data represent the first spectroscopic distinction, in the physiological temperature range, between O2 binding and electron transfer during the O2 reaction of mammalian cytochrome c oxidase. 6. A mechanism is presented for the O2 reaction of the mixed-valence state of cytochrome c oxidase involving four intermediate species. Electron transfer during this reaction is slow, relative to that seen with the fully reduced enzyme, and probably accounts for the detectability of the oxyferro species under these conditions.
Mechanistic insight into proton-coupled mixed valency