Atomistic insights into cardiolipin binding sites of cytochrome c oxidase.

Mitochondrial cytochrome c oxidase couples the reduction of oxygen to proton pumping. Despite an overall good understanding of its molecular mechanism, the role of cardiolipin in protein function is not understood. Here, we have studied the cardiolipin-protein interactions in a dynamic context by means of atomistic molecular dynamics simulations performed on the entire structure of monomeric and dimeric forms of the enzyme. Several microseconds of simulation data reveal that the crystallographic cardiolipin molecules that glue two monomers together bind weakly in hybrid and single-component lipid bilayers and dissociate rapidly. Atomistic simulations performed in the absence of tightly bound cardiolipin molecules strongly perturb the structural integrity of subunits III and VIIa, thereby highlighting an indispensable nature of lipid-protein interactions in enzyme function such as proton uptake and oxygen channeling. Our results demonstrate the strength of molecular simulations in providing direct atomic description of lipid-protein processes that are difficult to achieve experimentally.

The H channel is not a proton transfer path in yeast cytochrome c oxidase.

Cytochrome c oxidases (CcOs) in the respiratory chains of mitochondria and bacteria are primary consumers of molecular oxygen, converting it to water with the concomitant pumping of protons across the membrane to establish a proton electrochemical gradient. Despite a relatively well understood proton pumping mechanism of bacterial CcOs, the role of the H channel in mitochondrial forms of CcO remains debated. Here, we used site-directed mutagenesis to modify a central residue of the lower span of the H channel, Q413, in the genetically tractable yeast Saccharomyces cerevisiae. Exchange of Q413 to several different amino acids showed no effect on rates and efficiencies of respiratory cell growth, and redox potential measurements indicated minimal electrostatic interaction between the 413 locus and the nearest redox active component heme a. These findings clearly exclude a primary role of this section of the H channel in proton pumping in yeast CcO. In agreement with the experimental data, atomistic molecular dynamics simulations and continuum electrostatic calculations on wildtype and mutant yeast CcOs highlight potential bottlenecks in proton transfer through this route. Our data highlight the preference for neutral residues in the 413 locus, precluding sufficient hydration for formation of a proton conducting wire.