Date of Degree

5-2015

Document Type

Dissertation

Degree Name

Ph.D.

Program

Physics

Advisor(s)

Marilyn Gunner

Subject Categories

Biophysics

Keywords

proton affinity; proton loading site; proton transfer

Abstract

Cytochrome c oxidase (CcO) is a large trans-membrane protein, which is the final enzyme in the respiratory electron transport chain in mitochondria or aerobic bacteria. It implements proton pumping through the mitochondrial membrane against the electrochemical gradient, by utilizing the chemical energy released by reducing O2 to water. The active site of the chemical reaction is called the Binuclear Center (BNC) that is made up of heme a3, CuB, a Tyrosine residue and their ligands. The protein is reduced four times by electron from cytochromes c to reduce O2 and to generate four different BNC redox states step by step. In each reduction step a proton is delivered to the BNC and another proton is pumped across the protein to increase the trans-membrane proton gradient. In CcO, the pumped proton is firstly located in the proton loading site (PLS), and then is released out of the protein. In these processes, a high conserved Glutamate residue, plays an essential role on the proton translocation either to the BNC or the PLS.

In this thesis, Multi-Conformational Continuum Electrostatics (MCCE) and Molecular Dynamics (MD) are combined to study the proton affinity (pKa) of the high conserved Glutamate residue and the identity of the PLS. This Glutamate residue is located in a hydrophobic cavity in the protein, and the simulations show that the hydration of the cavity is controlled by the protonation state of the propionic acid of heme a3, a group on the proton outlet pathway. The changes in hydration and electrostatic interactions lower the proton affinity by at least 5 kcal/mol. The identity of the residues in the PLS is another open question in CcO research, and various groups above the BNC have been considered as candidates. We designed a new model for the simulation via separating the catalytic cycle into smaller substates and monitoring the charge of all residues in the protein. The results demonstrates the PLS is a cluster rather than a single residue, and the proton affinity of the heme a3 propionic acids primarily determines the number of protons loaded into the PLS.

Included in

Biophysics Commons

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