Localization of a quinol oxidase domain of the cytochrome d complex of Escherichia coli
Dueweke, Thomas Jerome
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Permalink
https://hdl.handle.net/2142/21643
Description
Title
Localization of a quinol oxidase domain of the cytochrome d complex of Escherichia coli
Author(s)
Dueweke, Thomas Jerome
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)
Chemistry, Biochemistry
Language
eng
Abstract
The aerobic respiratory chain of Escherichia coli contains two terminal oxidases, the cytochrome d complex and the cytochrome o complex. Each of these enzymes catalyzes the oxidation of ubiquinol-8 within the cytoplasmic membrane and the reduction of molecular oxygen to water. Both oxidases are coupling sites in the respiratory chain. The cytochrome d complex is a heterodimer (subunits I, II) which has three heme prosthetic groups. The hydropathy profiles of the amino acid sequences suggest that each subunit has multiple membrane-spanning helical segments. In this work, an effort to determine the topological folding of the two subunits across the membrane using $\beta$-galactosidase gene fusions is discussed, and a tentative topological model is proposed. Aspects of the model are confirmed in this work via proteolysis and antibody binding experiments. Previous studies characterized two monoclonal antibodies which bind to subunit I and specifically block the ability of the enzyme to oxidize ubiquinol. In this work, the epitopes of both these monoclonal antibodies have been mapped to within a single eleven amino acid stretch of subunit I. The epitope is located in a large, hydrophilic loop between the fifth and sixth putative membrane-spanning segments. Binding experiments with these monoclonal antibodies show this polypeptide loop to be periplasmic. Studies with proteolysis using trypsin and chymotrypsin also show the loop containing the antibody epitope to be periplasmic. An interesting effect of both the antibody binding and limited proteolysis with trypsin or chymotrypsin is the specific inhibition of the cytochrome d complex quinol oxidase activity. The ability of the complex to reduce oxygen using an alternative electron donor, N,N,N$\sp\prime$,N$\sp\prime$-tetramethyl-p-phenylene-diamine (TMPD), is not affected. Both the antibody binding and the proteolytic effects are localized to the same periplasmic loop that probably lies close to His186, a residue identified as one of the axial ligands of cytochrome b$\sb{558}$. Together, these data begin to define a functional domain where ubiquinol is oxidized near the periplasmic surface of the membrane at a site spatially separate from the site of oxygen reduction.
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