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|Title:||Determination of Structure-Function Relationships in the Cytochrome Bo Oxidase From Escherichia Coli|
|Author(s):||Thomas, Jeffrey William|
|Doctoral Committee Chair(s):||Gennis, Robert B.|
|Department / Program:||Chemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Cytochrome bo oxidase from Escherichia coli and the cytochrome c oxidases from bacteria, plants and animals belong to a superfamily of oxidases with a high degree of amino acid sequence homology and other structural similarities. The identifying structural feature of this oxidase superfamily is the presence of a heme-copper binuclear center. Functionally, these enzymes have in common the ability to carry out the four electron reduction of oxygen to water coupled concomitantly to the vectorial pumping of protons across the membrane.
In the work presented here, site-directed mutants in the cytochrome bo oxidase were constructed and then characterized to identify structure-function relationships within the enzyme. By analogy, one can apply these observations to all members of the heme-copper oxidase superfamily. All mutations were made in subunit I since this is the most highly conserved subunit among the heme-copper oxidases, and it contains all the metal centers in cytochrome bo. Fourier transform infrared spectroscopy, flow-flash kinetic measurements, and proton pumping activity measurements were employed to probe the oxidase mutants. FTIR results have shown residues in two highly conserved helices in subunit I are components of the binuclear center pocket, and a tyrosine residue may be a ligand to the binuclear center copper. Further results have identified an aspartic acid residue in an interhelical loop that can decouple electron transfer from proton pumping. Flow-flash kinetic measurements have also identified other residues that may be important for proton delivery to the binuclear center, the site of oxygen reduction.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1994.
|Date Available in IDEALS:||2014-12-17|