Time dependence of the catalytic intermediates in cytochrome c oxidase

Cytochrome c oxidase, the terminal enzyme in the electron transfer chain, c atalyzes the reduction of oxygen to water in a multiple step process by uti lizing four electrons from cytochrome c, To study the reaction mechanism, t he resonance Raman spectra of the intermediate states were measured during single turnover of the enzyme after catalytic initiation by photolysis of C O from the fully reduced GO-bound enzyme. By measuring the change in intens ity of lines associated with heme a, the electron transfer steps were deter mined and found to be biphasic with apparent rate constants of similar to 4 0 x 10(3) s(-1) and similar to 1 x 10(3) s(-1). The time dependence for the oxidation of heme a and for the measured formation and decay of the oxy, t he ferryl ("F"), and the hydroxy intermediates could be simulated by a simp le reaction scheme. In this scheme, the presence of the "peroxy" ("P") inte rmediate does not build up a sufficient population to be detected because i ts decay rate is too fast in buffered H2O at neutral pH. A comparison of th e change in the spin equilibrium with the formation of the hydroxy intermed iate demonstrates that this intermediate is high spin. We also confirm the presence of an oxygen isotope-sensitive line at 355 cm(-1), detectable in t he spectrum from 130 to 980 mu s, coincident with the presence of the F int ermediate.