The reaction of oxidized bovine heart cytochrome c oxidase (CcO) with one equivalent of hydrogen peroxide results in the formation of two spectrally distinct species. The yield of these two forms is controlled by the ionization of a group with a pK(a) of 6.6. At basic pH, where this group is deprotonated, an intermediate called P dominates (P, because it was initially believed to be a peroxy compound). At acidic pH where the group is protonated, a different species, called F (ferryl intermediate) is obtained. We previously proposed that the only difference between these two species is the presence of one proton in the catalytic center of F that is absent in P. It is now suggested that the catalytic center of this F form has the same redox and protonation state as a second ferryl intermediate produced at basic pH by two equivalents of hydrogen peroxide; the role of the second equivalent of H(2)O(2) is that of a proton donor in the conversion of P to F. Two chloride-binding sites have been detected in oxidized CcO. One site is located at the binuclear center; the second site was identified from the sensitivity of g=3 signal of cytochrome a to chloride in the EPR spectra of oxidized CcO. Turnover of CcO releases chloride from the catalytic center into the medium probably by one of the hydrophobic channels, proposed for oxygen access, with an orientation parallel to the membrane plane. Chloride in the binuclear center is most likely not involved in CcO catalysis. The influence of the second chloride site upon several reactions of CcO has been assessed. No correlation was found between chloride binding to the second site and the reactions that were examined.