Trypsin proteolysis of the cytochrome d complex of Escherichia coli selectively inhibits ubiquinol oxidase activity while not affecting N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity.
The cytochrome d complex is one of two membrane-bound terminal oxidases of the Escherichia coli aerobic respiratory chain. Previous studies have shown that this enzyme reconstituted into proteoliposomes rapidly oxidizes ubiquinol-8 as well as the soluble homologue, ubiquinol-1, and that quinol oxidase activity is accompanied by the formation of a transmembrane H+ electrochemical gradient. The enzyme also oxidizes the artificial reductant, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) with the generation of a H+ electrochemical gradient. In this work, it is established that trypsin digestion of the purified cytochrome d complex cleaves subunit I while subunit II is unaffected. Proteolysis of subunit I is correlated with loss of ubiquinol-8 and ubiquinol-1 oxidase activities. Trypsin digestion has no effect on TMPD oxidase activity. The cytochrome d complex is concluded to possess three distinct active sites for 1) ubiquinol oxidation, 2) TMPD oxidation, and 3) oxygen binding and reduction. Data also suggest that both sites of ubiquinol and TMPD oxidations are located on the periplasmic side of the E. coli membrane while the site of oxygen reduction is on the opposite side.
Lorence
R M
RM
Department of Chemistry, University of Illinois, Urbana 61801.
Carter
K
K
Gennis
R B
RB
Matsushita
K
K
Kaback
H R
HR
D000818:Animals; D017937:Cytochrome d Group; D003580:Cytochromes / Q000378:metabolism*; D003956:Dialysis; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D011808:Quinone Reductases / Q000037:antagonists & inhibitors*; D013772:Tetramethylphenylenediamine / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism
D003580:Cytochromes; D046911:Macromolecular Substances; D014451:Ubiquinone; D017937:Cytochrome d Group; D010089:Oxidoreductases, N-Demethylating; C022726:tetramethylphenylenediamine oxidase; D011808:Quinone Reductases; C030914:ubiquinol-1 oxidase; D014357:Trypsin; C003741:ubiquinol; D013772:Tetramethylphenylenediamine