Probing Gas Diffusion Pathways in Cytochrome C Oxidase with Explicit and Implicit Ligand Samplings

Abstract

Cytochrome C oxidase (CCO) couples reduction of O2 to water with proton pumping across the membrane, thereby generating a driving force for ATP synthesis. The x-ray structures of A-, B- and C-type CCOs suggest that an elongated hydrophobic cavity connecting the membrane core to the protein's active site might serve as an O2 access point to the catalytic active site of the enzyme. The structure of this cavity is, however, varied in different CCOs. While B- and C-type CCOs exhibit two entrances into this cavity, A-type CCOs appear to only have one entrance. To investigate the involvement of the hydrophobic cavity in O2 diffusion and to identify (potential) additional O2 entry pathways, we employed two complementary approaches using molecular dynamics simulations, performed on membrane-bound models of various CCO isoforms. In one approach, using a large ensemble of equilibrated protein conformations collected in the absence of O2, free-energy of O2 insertion over a grid covering the entire protein matrix is calculated using the “implicit ligand sampling” method. In the other approach, we included O2 molecules explicitly in the simulations and monitored their diffusion through the system. We observed favorable O2 binding and rapid O2 diffusion primarily from the membrane core, characterizing the hydrophobic cavity as a major O2 delivery pathway. Moreover, through simulations performed on a mutant enzyme, we identify a site that may contribute to the experimentally observed diffusion-controlled O2 binding kinetics in B-type CCO from Thermus thermophilus.