Cytochrome c oxidase, the terminal enzyme in the electron transfer chain, catalyzes the reduction of oxygen to water in a multiple step process by utilizing four electrons from cytochrome c. To study the reaction mechanism, the resonance Raman spectra of the intermediate states were measured during single turnover of the enzyme after catalytic initiation by photolysis of CO from the fully reduced CO-bound enzyme. By measuring the change in intensity of lines associated with heme a, the electron transfer steps were determined and found to be biphasic with apparent rate constants of ~40 x 103 s-1 and ~1 x 103 s-1. The time dependence for the oxidation of heme a and for the measured formation and decay of the oxy, the ferryl ('F'), and the hydroxy intermediates could be simulated by a simple reaction scheme. In this scheme, the presence of the 'peroxy' ('P') intermediate does not build up a sufficient population to be detected because its decay rate is too fast in buffered H2O at neutral pH. A comparison of the change in the spin equilibrium with the formation of the hydroxy intermediate demonstrates that this intermediate is high spin. We also confirm the presence of an oxygen isotopesensitive line at 355 cm-1, detectable in the spectrum from 130 to 980 μs, coincident with the presence of the F intermediate.