Computational investigation of oxygen reduction and proton pumping in cbb3-type Cytochrome c Oxidases

Heme-copper oxidases terminate the respiratory chain in many eukaryotes and prokaryotes as the final electron acceptors. They catalyze the reduction of molecular oxygen to water, and conserve the free-energy by proton pumping across the inner mitochondrial membrane or plasma membrane of bacteria. This leads to the generation of an electrochemical gradient across the membrane, which is utilized in the synthesis of ATP. The catalytic mechanism of oxidase is a complex coupling of electrons and protons, which has been studied with the help of numerous biophysical and biochemical methods. The superfamily of oxidases is classified into three different subfamilies; A-, Band C-type. The Aand B-type oxidases have been studied in great depth, whereas relatively less is known about the molecular mechanism of distinct C-type (or cbb3-type) oxidases. The latter enzymes, which are known to possess unusually high oxygen affinity relative to the former class of enzymes, also share little sequence or structural similarity with the Aand B-type oxidases. In the work presented in this thesis, C-type oxidases have been studied using a variety of computational procedures, such as homology modeling, molecular dynamics simulations, density functional theory calculations and continuum electrostatics. Homology models of the C-type oxidase correctly predicts the side-chain orientation of the cross-linked tyrosine and a proton-channel. The active-site region is also modelled with high accuracy in the models, which are subsequently used in the DFT calculations. With the help of these calculations it is proposed that the different orientation of the cross-linked tyrosine, and a strong hydrogen bond in the proximal side of the high-spin heme are responsible for the higher apparent oxygen affinity and a more rhombic EPR signal in the C-type oxidases. Furthermore, the pKa profiles of two amino acid residues, which are located close to the active-site, suggest a strong electron-proton coupling and a unique proton pumping route. Molecular dynamics simulations on the two-subunit C-type oxidase allowed for the first time to observe redox state dependent water-chain formation in the protein interior, which can be utilized for the redox coupled proton transfer.