Effective Pumping Proton Collection Facilitated by a Copper Site (CuB) of Bovine Heart Cytochrome c Oxidase,Revealed by a Newly Developed Time-resolved Infrared System

X-ray structural and mutational analyses have shown that bovine heart cytochrome c oxidase (CcO) pumps protons electrostatically through a hydrogen bond network using net positive charges created upon oxidation of a heme iron (located near the hydrogen bond network) for O₂ reduction. Pumping protons are transferred by mobile water molecules from the negative side of the mitochondrial inner membrane through a water channel into the hydrogen bond network. For blockage of spontaneous proton back-leak, the water channel is closed upon O₂ binding to the second heme (heme a₃) after complete collection of the pumping protons in the hydrogen bond network. For elucidation of the structural bases for the mechanism of the proton collection and timely closure of the water channel, conformational dynamics after photolysis of CO (an O₂ analog)-bound CcO was examined using a newly developed time-resolved infrared system feasible for accurate detection of a single C=O stretch band of α-helices of CcO in H₂O medium. The present results indicate that migration of CO from heme a₃ to Cu_B in the O₂ reduction site induces an intermediate state in which a bulge conformation at Ser-382 in a transmembrane helix is eliminated to open the water channel. The structural changes suggest that, using a conformational relay system, including Cu_B, O₂, heme a₃, and two helix turns extending to Ser-382, Cu_B induces the conformational changes of the water channel that stimulate the proton collection, and senses complete proton loading into the hydrogen bond network to trigger the timely channel closure by O₂ transfer from Cu_B to heme a₃.