Purification and Characterization of the Cytochrome-O Containing Terminal Oxidase of Escherichia Coli
Carter, Kimberly
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Permalink
https://hdl.handle.net/2142/70564
Description
Title
Purification and Characterization of the Cytochrome-O Containing Terminal Oxidase of Escherichia Coli
Author(s)
Carter, Kimberly
Issue Date
1987
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
The function of the aerobic respiratory chain of E. coli is to convert the energy released by the transfer of electrons from substrate molecules to oxygen into a proton gradient that can be used by the cell for ATP synthesis, locomotion and active transport of ions and molecules into and out of the cell.
One component of this chain is cytochrome o oxidase. This enzyme was purified from membranes of E. coli by a relatively simple purification procedure. The oxidase contains four subunits as identified by SDS-polyacrylamide gel $({\rm M\sb{r}}$ = 55, 33, 22 and 17). The enzyme contains 18 nmoles heme b mg$\sp{-1}$ of protein and 2 moles of iron and 2 moles of copper per mole of oxidase complex. Both irons are accounted for in cytochromes b $\sb{555}$ and b $\sb{562}$.
Cytochrome o utilizes both ubiquinol-1 and ubiquinol-8 as substrates. Activity is increased in the presence of phospholipids and detergents and activity is inhibited by cyanide, azide and HQNO. Proteoliposomes reconstituting the pyruvate: oxygen oxidoreductase chain, generate a proton electrochemical gradient (interior negative and alkaline) of 100 to 180 mV. The results presented here are consistent with this interpretation that the oxidase generates an electrical potential, interior negative, due to the vectorial electron flow from the outer to the inner surface of the membrane. The pH gradient, interior alkaline, results from scalar reactions that consume and release protons at the inner and outer surfaces of the membrane, respectively. Thus, cytochrome o does not appear to catalyze vectorial proton pumping.
The cytochrome o complex reduces oxygen to water with no measureable peroxide or superoxide as products. The terminal oxidase complex binds four molecules of carbon monoxides and, at present, the manner in which this occurs is unknown.
Monoclonal and polyclonal antibodies have been produced which recognize the oxidase. These antibodies are able to recognize cytochrome o and bind and inhibit the enzyme.
The cytochrome o terminal oxidase complex serves to transfer electrons from ubiquinol to oxygen in a relatively simple electron transport chain.
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