Structure-function relationship studies of the cytochrome bd oxidase of Escherichia coli
Ghaim, Joshua Eyassu B.
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https://hdl.handle.net/2142/23748
Description
Title
Structure-function relationship studies of the cytochrome bd oxidase of Escherichia coli
Author(s)
Ghaim, Joshua Eyassu B.
Issue Date
1996
Doctoral Committee Chair(s)
Gennis, Robert B.
Department of Study
Biology, Molecular
Chemistry, Biochemistry
Biophysics, General
Discipline
Biology, Molecular
Chemistry, Biochemistry
Biophysics, General
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Chemistry, Biochemistry
Biophysics, General
Language
eng
Abstract
The cytochrome bd oxidase complex is one of two terminal oxidases which are components of the aerobic respiratory chain of Escherichia coli. This membrane-bound oxidase catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water. Enzyme turnover generates proton and voltage gradients across the bilayer. The oxidase is a heterodimer containing three heme prosthetic groups, $b\sb{558},$ $b\sb{595},$ and d. To explain the functional properties of the oxidase, a simple model has been proposed in which the heme prosthetic groups define two separate active sites on opposite sides of the membrane at which the oxidation of ubiquinol (heme $b\sb{558})$ and the reduction of oxygen to water (hemes $b\sb{595}$ and d) take place. Two histidines, His19 and His186, both in subunit I have been shown to affect the incorporation of the hemes as well as the activity of the oxidase. His19 is proposed to be an axial ligand to either $b\sb{595}$ or d hemes, whereas His186 is believed to be one of the two axial ligands to heme $b\sb{558}.$
This thesis describes a combination of molecular biology, biophysical characterization and use of artificial proteases to try to understand the structure-function relationship of the cytochrome bd oxidase. A novel experiment is described in detail to show proximity relationships between sites on the surface of the two subunits of the oxidase. The artificial protease iron (S)-1- (p-(bromoacetamido)benzyl) -EDTA (Fe-BABE) was conjugated to selective reactive cysteines placed in subunits I and II and then activated with $\rm H\sb2O\sb2$ and ascorbate, and hydrolysis products were isolated and analyzed. Among other results, we found that residues 39 and 48 both of subunit II are within 12A of residues 255 and 337 of subunit I in the putative quinone-binding domain (Q-loop).
Site-directed mutagenesis and biophysical characterization were used: to construct a functional cytochrome bd oxidase devoid of cysteinyl residues, to try to determine the remaining ligands to the heme groups and also to identify residues that are involved in the oxidation and/or binding of ubiquinol. A successful construction of a cysteine-less oxidase is described in detail which appears to have the high-spin heme, heme $b\sb{595},$ being converted to a low-spin heme upon purification. Also described is some work done in collaboration with members of our laboratory in the determination of the remaining axial ligands to the heme groups. Met393 is proposed to be the second axial ligand to heme $b\sb{558}$ and His19 might be involved in the ligation of heme $b\sb{595}.$
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