The O2 reduction site of bovine cytochrome c oxidase (CcO) comprises two redox-active metal centers: Fe a3 and CuB. O2 is reduced at Fe a3 by four electrons transferred from cytochrome c in the P-side phase. Three proton-conducting pathways, D, K, and H, have been identified. Two distinct proton-pumping mechanisms, the D- and H-pathway mechanisms, proposed over 30 years ago, remain a subject of active debate. The former proposes that D-pathway transfers both pumping and water forming protons, whereas the latter proposes that the H-pathway transfers the pumping protons. CcOs are distributed across all aerobic organisms and are classified into evolutionarily related families: A, B, and C. In this study, we analyzed the common three-dimensional (3D) structural features of representative CcOs from each family to identify the proton-pumping system, assuming that the 3D structures responsible for the fundamental function of CcO, O2 reduction coupled with proton pumping, are evolutionarily conserved. Our analysis reveals that the 3D structural elements essential for proton pumping via the H-pathway mechanism are conserved across all three CcO families. These conserved elements include: 1) the site for loading and active release of pumping-protons to the P-side phase; 2) a water channel with a gate which opens to collect pumping protons from the N-side before the catalytic cycle starts and closes during the catalytic cycle to prevent leakage of pumping protons; 3) a water cluster located above the water-channel gate for storing pumping protons delivered from the water channel and transferring them in a timely manner to the proton-loading/release site when the gate is closed; and 4) a pumping-proton pool system, located below the water-channel gate, for the facile supply of protons to the water cluster when the gate opens. This structural conservation suggests that the H-pathway is responsible for proton pumping. Experimental results, supporting the D-pathway mechanism, do not disprove (are consistent with) the H-pathway mechanism. However, structural elements required to prevent pumping-proton leakage to the O2-reduction site and to the N-side surface, indispensable for the D-pathway as a proton pumping system, have not been identified experimentally.
Shimada et al. (Fri,) studied this question.