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|Title:||Molecular Studies of the NADH:ubiquinone Oxidoreductases and Thebo-Type Terminal Oxidase of the Aerobic Respiratory Chain of Escherichia Coli|
|Author(s):||Calhoun, Melissa White|
|Doctoral Committee Chair(s):||Gennis, Robert B.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The respiratory chains of aerobic organisms are composed of several membrane-bound proteins, which are responsible for the majority of energy production which occurs in living cells. Three such proteins, two NADH:ubiquinone oxidoreductases and a terminal ubiquinol oxidase, from the respiratory chain of the bacterium Escherichia coli are addressed in this thesis. These three enzymes were examined by a variety of genetic and spectroscopic techniques.
Although it had previously been speculated that E.coli contains two NADH:ubiquinone oxidoreductases, this thesis work demonstrates that the two biochemically-identified activities are genetically distinct. A site-directed mutant which inactivates one of the genetic loci, ndh, was constructed and the resulting bacterial strain maintains NADH:ubiquione oxidoreductase activity. The genetic map position of the locus, nuo, which encodes this second respiratory NADH:ubiquinone oxidoreductase, was also identified.
The major focus of this thesis is the identification of the protein residues which are ligands for the prosthetic groups of the bo-type terminal oxidase of E.coli. This enzyme is a member of a large superfamily of heme-copper oxidases which includes the $aa\sb3$-type oxidase of mammalian mitochondria. The ligands of both of the hemes of the E.coli oxidase were identified as histidine residues by electron paramagnetic spectroscopy. This knowledge, in combination with the identification of six invariant histidine residues in subunit I of the heme-copper oxidases, formed the basis for site-directed mutagenesis experiments. Mutant enzymes were characterized by optical, electron paramagnetic and Fourier transform infrared spectroscopies. The results of these studies have confirmed the previous identification of the two histidines which are ligands for one of the two heme groups. In addition, two residues which are essential for the binding of copper were also identified. A methionine residue which had previously been proposed to be a ligand for copper was found to be non-essential. Finally, one histidine residue was determined to be the ligand for the heme at the oxygen reduction site of the enzyme. These results, in combination with the analysis of the primary sequence of the protein by a variety of predictive methods, have been used in the creation a low-resolution three-dimensional model of the catalytic core of the heme-copper oxidases. This represents the first experimentally-based model of the active site of the heme-copper oxidases.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993.
|Date Available in IDEALS:||2014-12-17|