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Title:The AA3 Type Cytochrome C Oxidase From Rhodobacter Sphaeroides: Insights Into the Proton Pumping Mechanism From the N139D Mutation in the D-Channel
Author(s):Pawate, Ashtamurthy S.
Doctoral Committee Chair(s):Gennis, Robert B.
Department / Program:Biophysics and Computational Biology
Discipline:Biophysics and Computational Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biophysics, General
Abstract:Cytochrome c oxidase, an integral membrane protein, is the final enzyme of the electron transport chain. The two sides of the membrane are denoted as the N side (the cytoplasmic side in bacteria and the matrix side of mitochondria in eukaryotes) and the P side (the periplasmic side in bacteria and the intermembrane space of mitochondria in eukaryotes) and have different electrical properties. Cytochrome c oxidase catalyzes the highly exergonic reduction of oxygen to water and generates a transmembrane electrochemical proton gradient. This transmembrane gradient provides the proton-motive force that is used by ATP synthase to generate ATP from ADP and inorganic phosphate. The chemistry of the oxygen reduction reaction involves the uptake of electrons from the N side and protons from the P side of the membrane. This by itself is responsible for part of the transmembrane electrochemical gradient. However, cytochrome c oxidase captures the excess free energy released during the oxygen reduction reaction and pumps additional protons from the N side of the membrane to the P side of the membrane. This results in a substantial contribution to the transmembrane electrochemical gradient and thereby to the energetics of the cell. The molecular mechanism of proton pumping is not well understood. This thesis describes the creation of a mutant cytochrome c oxidase from Rhodobacter sphaeroides that retains complete oxygen reduction activity but has no proton pumping activity. This mutant oxidase, along with additional mutant oxidases with the same uncoupling mutation in the background, was studied with various biophysical techniques to gain an understanding of the observed phenotype. The work done in this thesis unravels the dependence of proton pumping on the pKa of E286 residue in subunit I of cytochrome c oxidase. The mutation N139D situated ∼20 A from E286 is shown to affect the p Ka of E286 and uncouples proton pumping from the oxygen reduction reaction.
Issue Date:2005
Description:151 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.
Other Identifier(s):(MiAaPQ)AAI3202152
Date Available in IDEALS:2015-09-25
Date Deposited:2005

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