X-ray structural analyses of azide-bound cytochrome c oxidases reveal that the H-pathway is critically important for the proton-pumping activity

Cytochrome c oxidase (CcO) is the terminal oxidase of cellular respiration, reducing O(2) to water and pumping protons. X-ray structural features have suggested that CcO pumps protons via a mechanism involving electrostatic repulsions between pumping protons in the hydrogen-bond network of a proton-conducting pathway (the H-pathway) and net positive charges created upon oxidation of an iron site, heme a (Fe(a)(2+)), for reduction of O(2) at another iron site, heme a(3) (Fe(a)(3)(2+)). The protons for pumping are transferred to the hydrogen-bond network from the N-side via the water channel of the H-pathway. Back-leakage of protons to the N-side is thought to be blocked by closure of the water channel. To experimentally test this, we examined X-ray structures of the azide-bound, oxidized bovine CcO and found that an azide derivative (N(3)(−)–Fe(a)(3)(3+), Cu(B)(2+)–N(3)(−)) induces a translational movement of the heme a(3) plane. This was accompanied by opening of the water channel, revealing that Fe(a)(3) and the H-pathway are tightly coupled. The channel opening in the oxidized state is likely to induce back-leakage of pumping protons, which lowers the proton level in the hydrogen-bond network during enzymatic turnover. The proton level decrease weakens the electron affinity of Fe(a), if Fe(a) electrostatically interacts with protons in the hydrogen-bond network. The previously reported azide-induced redox-potential decrease in Fe(a) supports existence of the electrostatic interaction. In summary, our results indicate that the H-pathway is critical for CcO's proton-pumping function.