Nye, Tacita Louise (2012) Dissection of the roles of individual terminal oxidases in aerobic respiration of Escherichia coli: the third oxidase, cytochrome bd II. PhD thesis, University of Sheffield.Full text not available from this repository. (Request a copy)
The terminal oxidases in Escherichia coli are essential for the functioning of the aerobic respiratory systems; they catalyse the terminal 4-electron reduction of oxygen to water at the expense of ubiquinol oxidation and contribute to the generation of a proton motive force. Currently, there are two well-understood terminal oxidases, cytochrome bd-I and cytochrome bo’. However, a third oxidase has been identified, cytochrome bd-II (AppBC). Despite its discovery over twenty years ago, very little work has been published on this elusive ubiquinol oxidase. Initial characterisation by others revealed that its subunit composition and spectral features are similar to those of cytochrome bd-I, but a clear function has not been elucidated. It was demonstrated in this laboratory that cytochrome bd-II gene expression is elevated in a strain lacking cytochrome bd-I and this evidence combined with earlier studies led to the suggestion that cytochrome bd-II may function as a non-proton-translocating electron sink during respiratory stress. However, the suggestion of a ‘non-electrogenic’ terminal oxidase has been controversial. More recent data demonstrated that cytochrome bd-II has an identical proton to electron ratio to cytochrome bd-I of 1. The aim of this study was to increase our understanding of cytochrome bd-II. Antibodies were produced using as antigens the terminal peptides of both subunits of each of the bd-type oxidases, with the aim of allowing their differentiation and comparison under varied levels of aerobiosis. Cytochrome bd-II showed maximal expression under anaerobic conditions, but its expression was elevated and apparently ‘unregulated’ in strains lacking cytochrome bd-I. The biochemical properties of cytochrome bd-II were also explored; with particular focus on its affinity for oxygen which was found to be much lower than that of cytochrome bd-I and similar to cytochrome bo’. Spectral and electrode-based studies demonstrated that cytochrome bd-I and cytochrome bd-II show a similar level of resistance to various respiratory inhibitors including cyanide and NO. Finally, the structural genes encoding cytochrome bd-II were cloned into a variety of plasmids and the conditions of growth for optimal protein expression were determined. The studies detailed herein have allowed further characterisation of cytochrome bd-II, but further research using the new tools developed is necessary to ensure a thorough understanding of this third terminal oxidase.
|Item Type:||Thesis (PhD)|
|Academic Units:||The University of Sheffield > Faculty of Science (Sheffield) > Molecular Biology and Biotechnology (Sheffield)|
|Depositing User:||Miss Tacita Louise Nye|
|Date Deposited:||29 Oct 2012 15:57|
|Last Modified:||08 Aug 2013 08:50|