Current Understanding on Cytochrome Bd Quinol Oxidase of Escherichia Coli: A Mutagenesis, Kinetics and Spectroscopic Study
Yang, Ke
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https://hdl.handle.net/2142/84869
Description
Title
Current Understanding on Cytochrome Bd Quinol Oxidase of Escherichia Coli: A Mutagenesis, Kinetics and Spectroscopic Study
Author(s)
Yang, Ke
Issue Date
2009
Doctoral Committee Chair(s)
Gennis, Robert B.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
Time-resolved kinetics study on the cytochrome bd quinol oxidase from Escherichia coli was carried out by stopped-flow techniques. The natural substrate, ubiquinol, was used to turnover the enzyme in the fast catalysis successfully for the first time. The results excluded the fully oxidized form of the enzyme from the rapid catalytic cycle of cytochrome bd oxidase. A re-investigation by both Flow-Flash and EPR on the previously reported mutant at Glu445 in subunit I, uncovered the dithionite-resistant ferric heme b595. Electrometrics data further suggested a series of protonatable groups forming a proton channel located in the membrane to facilitate the proton translocation from cytoplasm to the heme b595/heme d binuclear center. With help of the increasing database of available cytochrome bd oxidase sequences, site-directed mutagenesis studies were carried out on the highly conserved residues of the enzyme. Mutations on two highly conserved acidic residues in subunit I--Glu99 and Glu107 were characterized in detail. The glutamine substitution at Glu107 was managed to obtain the FTIR redox difference spectra regarding its relatively intact binuclear center. Glu107 was shown to be protonated at pH 7.6 and that it was perturbed by the reduction of the heme b595/heme d binuclear center at the active site. Mutations on Serine140, another structurally important non-acidic residue were also studied by FTIR. Mutant enzyme in which Serine140 was replaced with threonine was shown to perturb protonation of several acidic residues including Glu107 and/or Glu99. Due to their close proximity to the active site, Glu99, Glu107 and Ser140 were suggested to be part of the proton channel. Two highly conserved residues in the Q-loop of subunit I--Glu257 and Asp239 were also examined for their possible involvement in substrate binding. The FTIR difference spectra indicated a reorganization of binding environment caused by Glu257 mutant, while besides contributing to substrate binding, Asp239 was also believed to play a critical role in maintaining the redox potential of heme b595 in a right level that rapid catalysis can be achieved.
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