In order to study the role of subunits III and IV of the cytochrome c oxidase from P. denitrificans for electron and proton transfer, electrochemically induced FT-IR difference spectra of the two- and of the four-subunit enzyme have been compared. These spectra reflect the alterations in the protein upon electron and proton transfer. Since the spectra are essentially identical, they clearly indicate that the additional subunits III and IV do not contribute to the FT-IR difference spectra of the four-subunit oxidase. Subunits III and IV are thus not involved in the reorganization of the polypeptide backbone and of single amino acids upon electron transfer and coupled proton transfer observed in the difference spectra in addition to heme contributions. The subtle differences between the FT-IR difference spectra that are attributed to the influence of protein-protein interactions between the subunits are discussed.