Proteolysis of the cytochrome d complex with trypsin and chymotrypsin localizes a quinol oxidase domain.
The cytochrome d complex is a two-subunit, membrane-bound terminal oxidase in the aerobic respiratory chain of Escherichia coli. The enzyme catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water. Previous work demonstrated that the site for ubiquinol oxidation was selectively inactivated by limited proteolysis by trypsin, which cleaves at a locus within subunit I. This work is extended to show that a similar phenomenon is observed with limited chymotrypsin proteolysis of the complex. The cleavage patterns are similar whether one uses the purified oxidase in nondenaturing detergent or reconstituted in proteoliposomes or uses spheroplasts of E. coli as the substrate for the proteolysis. Hence, the protease-sensitive locus is periplasmic in the cell. Fragments resulting from proteolysis were characterized by N-terminal sequencing and by immunoblotting with the use of a monoclonal antibody of known epitope within subunit I. The data indicate that inactivation of the ubiquinol oxidase activity results from cleavage at specific residues with a hydrophilic region previously defined as the Q loop. This domain has been already implicated in ubiquinol oxidation by the use of inhibitory monoclonal antibodies. Electrochemical and HPLC analysis of the protease-cleaved oxidase suggests no global changes in either the quaternary or tertiary structure of the enzyme. It is likely that the Q loop is directly involved in forming a portion of the ubiquinol binding site near the periplasmic surface of the membrane.
Dueweke
T J
TJ
Department of Biochemistry, University of Illinois, Urbana 61801.
Gennis
R B
RB
D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D002918:Chymotrypsin / Q000494:pharmacology*; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D004789:Enzyme Activation / Q000187:drug effects; D000939:Epitopes; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D011487:Protein Conformation; D014357:Trypsin / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; D010088:Oxidoreductases; C020094:duroquinol oxidase; C040077:cytochrome bd terminal oxidase complex, E coli; D002918:Chymotrypsin; D014357:Trypsin