The three-dimensional structure of a proton-pumping pathway, the H-pathway, is evolutionarily conserved in all three families of cytochrome c oxidase

The O 2 reduction site of bovine cytochrome c oxidase (CcO) comprises two redox-active metal centers: Fe a 3 and Cu B . O 2 is reduced at Fe a 3 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, O 2 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 O 2 -reduction site and to the N-side surface, indispensable for the D-pathway as a proton pumping system, have not been identified experimentally.