The O
2
reduction site of cytochrome
c
oxidase (CcO), comprising iron (Fe
a
3
) and copper (Cu
B
) ions, is probed by x-ray structural analyses of CO, NO, and CN
-
derivatives to investigate the mechanism of the complete reduction of O
2
. Formation of the
derivative contributes to the trigonal planar coordination of
and displaces one of its three coordinated imidazole groups while a water molecule becomes hydrogen bonded to both the CN
-
ligand and the hydroxyl group of Tyr244. When O
2
is bound to
, it is negatively polarized (
), and expected to induce the same structural change induced by CN
-
. This structural change allows
to receive three electron equivalents nonsequentially from
,
, and Tyr-OH, providing complete reduction of O
2
with minimization of production of active oxygen species. The proton-pumping pathway of bovine CcO comprises a hydrogen-bond network and a water channel which extend to the positive and negative side surfaces, respectively. Protons transferred through the water channel are pumped through the hydrogen-bond network electrostatically with positive charge created at the Fe
a
center by electron donation to the O
2
reduction site. Binding of CO or NO to
induces significant narrowing of a section of the water channel near the hydrogen-bond network junction, which prevents access of water molecules to the network. In a similar manner, O
2
binding to
is expected to prevent access of water molecules to the hydrogen-bond network. This blocks proton back-leak from the network and provides an efficient gate for proton-pumping.