Pyruvate Oxidase and the Cytochrome D Terminal Oxidase of Escherichia Coli
Koland, John George
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https://hdl.handle.net/2142/70223
Description
Title
Pyruvate Oxidase and the Cytochrome D Terminal Oxidase of Escherichia Coli
Author(s)
Koland, John George
Issue Date
1983
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Physical
Abstract
Two purified components of the Escherichia coli aerobic electron transport system were studied, pyruvate oxidase and the cytochrome d terminal oxidase. Three general subjects were treated: (i) the active site structure and catalytic mechanism of pyruvate oxidase, (ii) the role of pyruvate oxidase in the E. coli aerobic electron transport system, and (iii) physical characteristics of the cytochrome d terminal oxidase.
The active site structure of pyruvate oxidase was investigated through chemical modification experiments. Application of reagents reacting specifically with arginine and cysteine residues showed that both of these amino acids were involved in the binding of the cofactor thiamin pyrophosphate. Reagents forming fluorescent cysteine conjugates were subsequently used in studies of the active site geometry. The distance between the thiamin pyrophosphate binding site and the flavin coenzyme was estimated by application of the fluorescence energy transfer technique.
The role of pyruvate oxidase in E. coli electron transport was studied by several means. E. coli membrane preparations were supplemented with purified oxidase and pyruvate-driven oxygen consumption monitored. The involvement of ubiquinone in electron transport from pyruvate to oxygen was demonstrated by use of membranes from quinone deficient strains of E. coli. The ubiquinone reductase activity of pyruvate oxidase was also characterized. In particular it was shown that pyruvate oxidase could reduce ubiquinone in a phospholipid environment. Finally, a functional electron transport system was reconstituted from purified components, including pyruvate oxidase, ubiquinone and the cytochrome d terminal oxidase.
Combined spectroscopic and electrochemical methods were applied in the physical characterization of the cytochrome d terminal oxidase. Through potentiometric analyses, the spectra of the individual heme components were resolved. Coulometric analyses yielded estimates for the absolute quantities of the heme components present in the oxidase.
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