Characterization of the cytochrome d terminal oxidase complex of Escherichia coli using polyclonal and monoclonal antibodies.
The cytochrome d terminal oxidase complex is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli. Previous work has shown by dodecyl sulfate-polyacrylamide gel electrophoresis that this enzyme contains two subunits (I and II) and three cytochrome components, b558 , a1, and d. Reconstitution studies have demonstrated that the enzyme functions as a ubiquinol-8 oxidase and catalyzes an electrogenic reaction, i.e. turnover is accompanied by a charge separation across the membrane bilayer. In this paper, monoclonal and polyclonal antibodies were used to obtain structural information about the cytochrome d complex. It is shown that antibodies directed against subunit I effectively inhibit ubiquinol-1 oxidation by the purified enzyme in detergent, whereas antibodies which bind to subunit II have no effect on quinol oxidation. The oxidation rate of N,N,N',N'-tetramethyl-p-phenylenediamine, in contrast, is unaffected by antisubunit I antibodies, but is inhibited by antibodies against subunit II. It is concluded that the quinol oxidation site is on subunit I, previously shown to be the cytochrome b558 component of the complex, and that N,N,N',N'-tetramethyl-p-phenylenediamine oxidation occurs at a secondary site on subunit II. The antibodies were also used to analyze the results of a protein cross-linking experiment. Dimethyl suberimidate was used to cross-link the subunits of purified, solubilized oxidase. Immunoblot analysis of the products of this cross-linking clearly indicate that subunit II probably exists as a dimer within the complex. Finally, it is shown that the purified enzyme contains tightly bound lipopolysaccharide. This was revealed after discovering that one of the monoclonal antibodies raised against the purified complex is actually directed against lipopolysaccharide. The significance of this finding is not known.
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D010088:Oxidoreductases / Q000032:analysis* / Q000276:immunology; D010446:Peptide Fragments / Q000032:analysis; D011808:Quinone Reductases / Q000037:antagonists & inhibitors; D013772:Tetramethylphenylenediamine / Q000378:metabolism
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010088:Oxidoreductases; D011808:Quinone Reductases; C030914:ubiquinol-1 oxidase; C040077:cytochrome bd terminal oxidase complex, E coli; D013772:Tetramethylphenylenediamine