Computational studies of proton pumping mechanism and water transport in cytochrome c oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory electron transport chain, which pumps protons across the inner mitochondrial or bacterial plasma membrane using energy derived from the reduction of O2 to H2O. Over ninety percent of biological reduction of atmospheric oxygen on Earth is accomplished by CcO present in various aerobic organisms. More than three decades since the identification of the true function of the enzyme and almost fifteen years since the first determination of the structure the protein have passed, yet its proton pumping mechanism is still not completely understood. This thesis presents computational studies that have been performed to gain insights into some of the remaining mysteries of the enzymatic mechanisms of CcO. Specifically, we focus on understanding the recent experimental data on the membrane potential generated by the enzyme, and possible proton pumping mechanisms that can explain such data. A closely related issue to proton translocation by the enzyme is the transport property of internal water molecules in CcO, which is the second major subject of this thesis.;In chapter 5, we discuss possible gating mechanisms of proton transfer in CcO, which guarantee that the pumped proton transfer occurs before the chemical proton transfer. We propose a new mechanism by which the hydrophobicity of the cavity near the catalytic center of the enzyme can regulate the number and dynamics of the water molecules in the cavity, thereby providing a way for proton gating.;Another fundamental question of the mechanism of CcO addressed in this work, in chapter 4, is related to the pathways and the mechanism of transport of water produced by the enzyme. Identification of the water exit pathways is crucial for understanding the proton pumping mechanism of CcO, because it can provide insights on the proton translocation pathways. With the aid of topological information on the internal cavities, obtained with our own new algorithm (the detail of which is described in chapter 2), we performed molecular dynamics simulations and PMF calculations to investigate the transport of internal water molecules in the enzyme; we have found two water exit pathways that appear to be conserved among different (eukaryotic and prokaryotic) species. (Abstract shortened by UMI.);One of the key features in the majority of currently discussed mechanisms of proton pumping by CcO is that the transfer of the pumped proton always precedes the transfer of the chemical proton used for the reduction of oxygen by the enzyme. In chapter 3, using an analysis that integrates the membrane potential obtained from the numerical solutions of the Poisson-Boltzmann equation and the kinetic data from the time-resolved potentiometry experiments, we examine this key assumption of the proposed mechanisms.