QM/MM analysis of redox driven proton pumping

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Cytochrome COxidase (CeO) is an important enzyme involved in the electron transfer pathway in cellular respiration. CeO activates molecular oxygen to prevent the release of potentially toxic oxygen intermediates and at the same time, uses the free energy from oxygen reduction to pump protons across the membrane in a stoichiometric fashion, creating the proton concentration gradient required for ATP synthesis. Compromise of CeO activity is implicated in serious human health threats such as various neurodegenerative diseases, muscular dystrophies and colon cancer. Therefore, investigation of the functional mechanism of CeO at a molecular level not only has fundamental value but also contributes to understanding the molecular origin of these diseases and aiding the design of effective therapeutic strategies. In this project, we propose to use quantum mechanical/molecular mechanical (QM/MM) techniques to study several actively debated mechanistic issues in CeO. Specifically, we have the following aims: (i). Use pKa calculations with QM/MM methods to help identify the most likely candidate(s) for the loading site of pumped protons among His334 and the propionates of the heme co-factor. (ii). Via analysis of the energetics (including barrier) of Glu286 isomerization and key proton transfer steps in wild type CeO, establish the identity of the "gating element(s)" that prevent the backflow of protons. Define conformational and electrostatic contributions to gating. Together with experimental analyses, these investigations at the atomic level will firmly establish the molecular properties of CeO that are essential to its function. Continued development of an approximate density functional approach will find application in the study of a broad range of biomolecules, especially those involve vectorial chemistry.