Molecular biology studies on thecyd operon of Escherichia coli
Fang, Hong
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Permalink
https://hdl.handle.net/2142/20946
Description
Title
Molecular biology studies on thecyd operon of Escherichia coli
Author(s)
Fang, Hong
Issue Date
1990
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)
Biology, Molecular
Biology, Microbiology
Chemistry, Biochemistry
Language
eng
Abstract
The Cytochrome d terminal oxidase complex is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli. The enzyme is located in the cytoplasmic membrane where it oxidizes ubiquinol-8 and reduce oxygen to water. The enzyme is an $\alpha\beta$ hetero-dimer containing hemes $b\sb{558}$, $b\sb{595}$ and d. The cyd locus, which encodes both subunits, has been cloned and mapped genetically. The thesis research described here is focused on further characterization of the cyd gene locus and on the structure/function relationship of the cytochrome d complex. Deletion and insertion analyses combined with in vitro coupled transcription and translation studies indicate that cyd is a two-cistron operon with the subunit I gene upstream of the subunit II gene. S1 nuclease mapping experiments were used to show that the initiation of transcription occurs at $-$287 bases upstream of the start codon of the subunit I gene. In order to define the heme binding sites, each of ten histidine residues within the two subunits were converted to other amino acids using site-directed mutagenesis. Spectroscopic studies and catalytic assays strongly suggest that his19 and his186 within subunit I are essential for heme binding and oxidase activity. His186 is an axial ligand for cytochrome $b\sb{558}$. His19 is an axial ligand for either cytochrome d or $b\sb{595}$. It is difficult to distinguish between these two hemes because the binding of the two hemes are always cooperative. These models are confirmed by revertant analyses. Finally the cydC gene locus is required for the activity of the cytochrome d complex.
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