Spectroscopic analysis and dynamics of ligand binding to bacterial oxidases
Hill, John Joseph
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Permalink
https://hdl.handle.net/2142/21061
Description
Title
Spectroscopic analysis and dynamics of ligand binding to bacterial oxidases
Author(s)
Hill, John Joseph
Issue Date
1994
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
Biophysics, General
Language
eng
Abstract
The aerobic respiratory chain of Escherichia coli contains two terminal oxidases, the cytochrome bd complex and the cytochrome bo complex. Each of these enzymes functions as a ubiquinol oxidase and reduces molecular oxygen to water. Although the two enzymes perform the same function they do not show any obvious similarity between their gene sequences and therefore, their structure. This thesis addresses the differences between these two enzymes in terms of the types and number of prosthetic groups and their reactions with ligands, as determined by a variety of spectroscopic methods.
Fourier transform infrared spectroscopy of CO bound to the enzymes was used to probe the environment of the heme-pocket, where oxygen is reduced to water, for each enzyme. The cytochrome bd complex was shown to contain a heme-heme binuclear center consisting of heme $b\sb{595}$ and heme d, that there is only one heme d per complex, and that the pocket environment is very hydrophobic. At cryogenic temperatures and following flash photolysis, the rate of relaxation of CO to heme d from the pocket surface follows the power law in time and is temperature dependent. At cryogenic temperatures and in the dark, CO is shown to migrate from heme d to heme $b\sb{595}$ and that this rate is not dependent upon temperature. The cytochrome bo complex was shown to contain a single copper atom per complex and that this copper atom forms a binuclear center with heme o, similar to that of the mitochondrial cytochrome c oxidase.
Electron paramagnetic resonance and visible spectroscopies, as well as, stopped-flow methods and equilibrium titrations were conducted for the reactions of cyanide and azide with the cytochrome bd complex. The results show that these ligands bind predominantly to heme d, and that the cytochrome bd complex's low sensitivity towards these classic respiratory inhibitors is due to their very slow association rate with heme d. Stopped-flow and flow-flash methods were employed to determine the affinity of O$\sb2$ and CO for the cytochrome bd complex. The affinity of O$\sb2$ for the cytochrome bd complex (K$\sb{\rm D}$ = 26 nM) is higher than for most other heme proteins. Furthermore, the O$\sb2$ affinity is similar to that of CO (K$\sb{\rm D}$ = 22 nM). These results represent the first detailed and systematic study of ligand binding to this enzyme, and provide a foundation for the study of mutant forms of the complex.
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