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Title:Studies on the structure, function and mechanisms of the cbb3 type cytochrome c oxidase from Vibrio cholerae and the cytochrome bo3 ubiquinol oxidase from Escherichia coli
Author(s):Ouyang, Hanlin
Director of Research:Gennis, Robert B.
Doctoral Committee Chair(s):Gennis, Robert B.
Doctoral Committee Member(s):Lu, Yi; Crofts, Antony R.; Wraight, Colin A.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Aurachin C1-10 synthesis
Vibrio cholerae cbb3 cytochrome c oxidase
proton pathway
redox inactive calcium
E.coli bo3 ubiquinol oxidase
Pm and F intermediates
tyrosine radical
Electron paramagnetic resonance (EPR)
unnatural amino acid
Abstract:Cytochrome c oxidases (CcO) are terminal oxidases in the respiratory chain and contribute to a large fraction in the heme-copper oxidase superfamily. They are transmembrane protein complexes catalyzing the reduction of dioxygen to water with a variety range of electron donors. During the process, electrons are transferred into the enzyme while protons are up taken at the same time. Some of the protons participate in the transformation of dioxygen into water as substrate. The rest of the protons are translocated across the plasma membrane, conserving energy in the form of electrochemical potential. Different subfamilies of CcO are identified mainly based on the sequence homology. They vary in a wide range of aspects but also share certain conserved features: a) A binuclear center consisted of CuB and a high spin heme for catalysis; b) a low spin heme and six histidines as metal ligand; c) at least one proton uptake pathway and d) catalytic cycle for dioxygen reduction. In this study, cbb3 type cytochrome c oxidase from V. cholerae and bo3 type ubiquinol oxidase from E. coli were investigated separately. Site directed mutagenesis were carried out in the case of cbb3 protein based on crystal structure and sequence alignment. Activity data suggested that the D pathway analogue is blocked with hydrophobic residues and no mutant altered enzymatic activity. Only one proton pathway exists which is analogous to K pathway in A type oxidases. Moreover, EIII49 from the third subunit were demonstrated to be most likely the entrance of K pathway. Two key residues E126 and E129 had been believed to be part of proton exit pathway. However, crystal structure, activity assay and metal analysis suggested the two residues are more than likely to be of structural importance rather than part of a proton pathway. A group of residues located in the proximity of heme b propionate groups were also studied and shown their correlation with subunits assembly and heme c incorporation. Bo3 type ubiquinol oxidase enzyme activity can be inhibited by quinol analogues. To facilitate the study of quinone binding sites of bo3, a competitive inhibitor aurachin 1-10 was synthesized through a seven-step synthetic route as my preliminary work on bo3 project. When mixed-valence cytochrome c oxidase (R2) reacts with dioxygen, the first adduct intermediate (A) decays into a radical intermediate (PM). It’s a four electron reduction of O2 in which two electrons comes from Fe2+, another from Cu+ and the source of fourth electron is still under debate. Injection of the first electron from electron donor will transform this PM intermediate into another one called F. In this work we studied the PM of bo3 protein by a mimic reaction with hydrogen peroxide. The time scale for the mimic reaction is at least 1000 times slower than the O2 steady state turn over which allowed a successful capture in detail the formation of PM and F. Mutant data suggested that Y288 is required for PM formation and Y173 is crucial for F formation. CW-EPR experiments demonstrated WT and Y288H generated a radical of the same origin while Y173 gave rise to a different radical species and Y288H+Y173F failed to generate radical of any kind. Auxotrophic strains were made for every mutant. We found out that when ring deuterated tyrosine (d4-tyr) was incorporated in to bo3, all the above radical signal collapsed into a simpler splitting pattern, which demonstrated the radicals originates on tyrosines. Further analysis of the signal splitting pattern revealed that the radical is on Y173 in the case of WT and Y288H, while in the case of Y173F radical is likely on Y288. Together with stop flow data, we were able to conclude that for H2O2 reaction, radical is first produced on the cross-linked Y288 to form PM and then migrate to Y173, for the decay of PM into F. We also took advantage of the amber codon suppressor technique developed by Peter Schultz lab and genetically replaced Y288 and Y173 with 3-amino-tyrosine (YNH2). The midpoint potential of YNH2 is 90mV lower than tyrosine and thus could serve as a potential radical trap. Characterization of Y288YNH2 and Y173YNH2 mutants were carried out in this thesis.
Issue Date:2013-02-03
Rights Information:Copyright 2012 Hanlin Ouyang
Date Available in IDEALS:2013-02-03
Date Deposited:2012-12

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