Abstract
Cytochrome c oxidase (CcO) reduces O(2) to water, coupled with a proton-pumping process. The structure of the O(2)-reduction site of CcO contains two reducing equivalents, Fe (a)(3)(2+) and Cu(B)(1+), and suggests that a peroxide-bound state (Fe (a)(3)(3+)-O(-)-O(-)-Cu(B)(2+)) rather than an O(2)-bound state (Fe (a)(3)(2+)-O(2)) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fe (a)(3)(2+)-O(2), whereas Fe (a)(3)(3+)-O(-)-O(-)-Cu(B)(2+) 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 (a)(3)(4+) = O(2-), not as Fe (a)(3)(4+)-OH(-) The distance suggests an ∼800-cm(-1) 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 (a)(3)(3+)-O(-)-O(-)-Cu(B)(2+) 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.