Cytochrome c oxidase (CcO) is the terminal oxidase of cell respiration which reduces molecular oxygen (O₂) to H2O coupled with the proton pump. For elucidation of the mechanism of CcO, the three-dimensional location and chemical reactivity of each atom composing the functional sites have been extensively studied by various techniques, such as crystallography, vibrational and time-resolved electronic spectroscopy, since the X-ray structures (2.8 Å resolution) of bovine and bacterial CcO have been published in 1995.X-ray structures of bovine CcO in different oxidation and ligand binding states showed that the O₂reduction site, which is composed of Fe (heme a 3) and Cu (CuB), drives a non-sequential four-electron transfer for reduction of O₂to water without releasing any reactive oxygen species. These data provide the crucial structural basis to solve a long-standing problem, the mechanism of the O₂reduction.Time-resolved resonance Raman and charge translocation analyses revealed the mechanism for coupling between O₂reduction and the proton pump: O₂is received by the O₂reduction site where both metals are in the reduced state (R-intermediate), giving the O₂-bound form (A-intermediate). This is spontaneously converted to the P-intermediate, with the bound O₂fully reduced to 2 O²⁻. Hereafter the P-intermediate receives four electron equivalents from the second Fe site (heme a), one at a time, to form the three intermediates, F, O, and E to regenerate the R-intermediate. Each electron transfer step from heme a to the O₂reduction site is coupled with the proton pump.X-ray structural and mutational analyses of bovine CcO show three possible proton transfer pathways which can transfer pump protons (H) and chemical (water-forming) protons (K and D). The structure of the H-pathway of bovine CcO indicates that the driving force of the proton pump is the electrostatic repulsion between the protons on the H-pathway and positive charges of heme a, created upon oxidation to donate electrons to the O₂reduction site. On the other hand, mutational and time-resolved electrometric findings for the bacterial CcO strongly suggest that the D-pathway transfers both pump and chemical protons. However, the structure for the proton-gating system in the D-pathway has not been experimentally identified. The structural and functional diversities in CcO from various species suggest a basic proton pumping mechanism in which heme a pumps protons while heme a 3 reduces O₂as proposed in 1978.