X-ray structures of catalytic intermediates of cytochrome c oxidase provide insights into its O 2 activation and unidirectional proton-pump mechanisms.

Cytochrome c oxidase (CcO) reduces O to water, coupled with a proton-pumping process. The structure of the O-reduction site of CcO contains two reducing equivalents, Fe and Cu, and suggests that a peroxide-bound state (Fe-O-O-Cu) rather than an O-bound state (Fe-O) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fe-O, whereas Fe-O-O-Cu is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe-O distance of the ferryl center could best be described as Fe = O, not as Fe-OH. The distance suggests an ∼800-cm Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fe-O-O-Cu state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.