The mechanism of dioxygen reduction catalyzed by cytochrome c oxidase, the terminal enzyme of the respiration chain of aerobic organisms, has been investigated with time-resolved resonance Raman spectroscopy. Five oxygen isotope sensitive Raman bands have been identified for O-16(2)/O-18(2) intermediates at 571/544, 804/764, 356/342, 785/750, and 450/425 cm(-1) in the order of appearance. The first and last ones, which have been assigned to the Fe-III-O-2(-) and Fe-III-OH- stretching modes, respectively, are in general consensus, but the remaining three bands are currently under debate. This study establishes that the 804/764 cm(-1) species is generated prior to the 785/750 cm(-1) species, and the latter is not observed when we start from the mixed valence enzyme. Therefore, the enzyme oxidation state of the 804/764 cm(-1) species is higher by one oxidative equivalent than that of the 785/750 cm(-1) species, although both bands have been assigned to the Fe=O stretching mode. The excitation wavelength dependences of these two sets of Raman bands are distinctly different, suggesting that the electronic properties of the two hemes are quite different. We found that the conversion from the 804/764 cm(-1) species to the 785/750 cm(-1) species was significantly slower in D2O than in H2O, suggesting strong coupling between electron and vector cm proton transfers at this stage. Presumably, Cu-B at the binuclear site causes heterolytic cleavage of the O-O bond, giving rise to an oxoiron in the Fe-v oxidation level of the enzyme with the 804/764 cm(-1) bands, and the electron transfer to this oxoiron is accompanied by some protein conformational changes, which cause distortion of the oxoheme and thus appearance of the His-Fe=O bending Raman bands at 356/342 cm(-1) and simultaneously active transport of protons.