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Title:Structural and Functional Studies of the Cytochrome D Oxidase Complex of Escherichia Coli
Author(s):Zuberi, Tamma Marie
Doctoral Committee Chair(s):Gennis, Robert
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Molecular
Biophysics, General
Abstract:The cytochrome d oxidase complex is one of two terminal quinol oxidase complexes of Escherichia coli. The heterodimeric complex contains three heme prosthetic groups, $b\sb{558}, b\sb{595},$ and d. Two histidines, His19 and His186, in subunit I are essential for retaining the heme groups. His19 was proposed to be an axial ligand to either $b\sb{595}$ or d, and His186 was postulated to be a ligand to low spin $b\sb{558}.$
This thesis describes a combination of site-directed mutagenesis and biophysical characterization to try to determine the remaining ligands to the heme groups. Electron nuclear double resonance spectroscopy indicates that the d heme does not have a histidine axial ligand. Additional mutations were made at His19, as well as in surrounding residues, in an attempt to more subtly alter the heme pocket. The results show that His19 is very important structurally, although it may not act as an axial ligand to either $b\sb{595}$ or d.
Resonance Raman spectroscopy showed $b\sb{558}$ spectra that were consistent with bishistidine ligation. However, all histidines had been eliminated as candidates by mutagenesis, and MCD results indicated His-Met ligation. Therefore, methionines within subunit I were mutated to determine the second axial ligand to heme$b\sb{558}$. Although none of the methionines were essential for the retention of function of the complex, the mutation Met393 to leucine causes $b\sb{558}$ to convert to a high-spin heme with altered ligand binding properties. Met393 is proposed to be the second axial ligand to heme $b\sb{558}.$
Two stable intermediates of the catalytic oxygen-reducing cycle of the complex, an Fe-O$\sb2$ and an oxo-ferryl species, were identified using resonance Raman spectroscopy. A catalytic scheme for oxygen reduction similar to that of cytochrome c oxidase is proposed. At the other active site, that of quinol oxidation, we have utilized electron paramagnetic resonance to detect and characterize a stable semiquinone radical of the cycle, which verifies the mechanism of single sequential electron transfer from the quinol to the initial electron acceptor of the complex, most probably the $b\sb{558}$ heme.
Issue Date:1993
Description:335 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993.
Other Identifier(s):(UMI)AAI9329212
Date Available in IDEALS:2014-12-17
Date Deposited:1993

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