Proton translocation in cytochrome c oxidase: Insights from proton exchange kinetics and vibrational spectroscopy

Abstract

Cytochrome c oxidase is the terminal enzyme in the electron transfer chain. It reduces oxygen to water and harnesses the released energy to translocate protons across the inner mitochondrial membrane. The mechanism by which the oxygen chemistry is coupled to proton translocation is not yet resolved owing to the difficulty of monitoring dynamic proton transfer events. Here we summarize several postulated mechanisms for proton translocation, which have been supported by a variety of vibrational spectroscopic studies. We recently proposed a proton translocation model involving proton accessibility to the regions near the propionate groups of the heme a and heme a3 redox centers of the enzyme based by hydrogen/deuterium (H/D) exchange Raman scattering studies (Egawa et al., PLOS ONE 2013). To advance our understanding of this model and to refine the proton accessibility to the hemes, the H/D exchange dependence of the heme propionate group vibrational modes on temperature and pH was measured. The H/D exchange detected at the propionate groups of heme a3 takes place within a few seconds under all conditions. In contrast, that detected at the heme a propionates occurs in the oxidized but not the reduced enzyme and the H/D exchange is pH-dependent with a pKa of ~8.0 (faster at high pH). Analysis of the thermodynamic parameters revealed that, as the pH is varied, entropy/enthalpy compensation held the free energy of activation in a narrow range. The redox dependence of the possible proton pathways to the heme groups is discussed.

The subunit composition and function of mammalian cytochrome c oxidase

“…A further mitochondrial disease, manifesting with encephalomyopathy, hydrocephalus and hypertrophic cardiomyopathy due to a missense p.R20C mutation in the COX6B1 gene, coding for COX subunit VIb-1, was recently described (Abdulhag et al, 2015). Finally, another case of a mitochondrial disease based on mutations in the nuclearcoded gene for COX subunit VIIb was described by Ishigami et al (2015).…”

“…Several recent review articles describe the known data and hypotheses of proton pumping in COX (Ferguson-Miller et al, 2012;Ishigami et al, 2015;Popović, 2013;Rich and Maréchal, 2013;Yoshikawa et al, 2011). Two proton pathways, the D and the K channels have been identified in COX from bacteria and mammals (Hellwig et al, 1998;Michel et al, 1998).…”

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

“…Because the catalytic sites of the heme-copper oxidases from eukaryotic and prokaryotic species share the same structural architecture, it has been proposed that the proton pumping mechanism is conserved across species (38,39). However, this general concept has been questioned by the lack of agreement between those mechanisms shown to be operative in bacterial CcOs (5, 6) and those proposed for bCcO (1,4,28,31). Illustrative of the distinct properties of the different species is the finding that although a structural change in helix-X, similar to that in bCcO, is seen in Rhodobacter sphaeriodes CcO (RsCcO) (29), the OH group of the heme a farnesyl side chain does not undergo the 160°rotation upon the change in the redox state of heme a as it does in bCcO.…”

“…S8). The allosteric structural transition establishes a dynamic communication pathway between the two hemes, offering structural insights into previously proposed proton translocation mechanisms in bCcO (1,4,28,31).…”

“…1), where oxygen is reduced to water by using four protons (the substrate protons) from the matrix side of the membrane and four electrons delivered to CcO by cytochrome c. Associated with the oxygen reduction chemistry, four additional protons (the pumped protons) are translocated from the negative side to the positive side of the mitochondrial membrane. Whereas the mechanism of the O 2 reduction is relatively well understood, the mechanism by which the redox energy is coupled to proton translocation remains unresolved in mammalian CcOs (1)(2)(3)(4), although it is quite well-defined in bacterial CcOs (5,6).…”