Reconstitution of the membrane-bound, ubiquinone-dependent pyruvate oxidase respiratory chain of Escherichia coli with the cytochrome d terminal oxidase.
Pyruvate oxidase is a flavoprotein dehydrogenase located on the inner surface of the Escherichia coli cytoplasmic membrane and coupled to the E. coli aerobic respiratory chain. In this paper, the role of quinones in the pyruvate oxidase system is investigated, and a minimal respiratory chain is described consisting of only two pure proteins plus ubiquinone 8 incorporated in phospholipid vesicles. The enzymes used in this reconstitution are the flavoprotein and the recently purified E. coli cytochrome d terminal oxidase. The catalytic velocity of the reconstituted liposome system is about 30% of that observed when the flavoprotein is reconstituted with E. coli membranes. It is also shown that electron transport from pyruvate to oxygen in the liposome system generates a transmembrane potential of at least 180 mV (negative inside), which is sensitive to the uncouplers carbonyl cyanide p-(tri-chloromethoxy)phenylhydrazone and valinomycin. A trans-membrane potential is also generated by the oxidation of ubiquinol 1 by the terminal oxidase in the absence of the flavoprotein. It is concluded that (1) the flavoprotein can directly reduce ubiquinone 8 within the phospholipid bilayer, (2) menaquinone 8 will not effectively substitute for ubiquinone 8 in this electron-transfer chain, and (3) the cytochrome d terminal oxidase functions as a ubiquinol 8 oxidase and serves as a "coupling site" in the E. coli aerobic respiratory chain. These investigations suggest a relatively simple organization for the E. coli respiratory chain.
D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group / Q000378:metabolism; D003580:Cytochromes; D004579:Electron Transport; D045222:Electron Transport Chain Complex Proteins; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008051:Lipid Bilayers; D008564:Membrane Potentials; D010088:Oxidoreductases / Q000378:metabolism*; D011771:Pyruvate Oxidase / Q000378:metabolism*; D014451:Ubiquinone / Q000378:metabolism
D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D014451:Ubiquinone; C030778:ubiquinone 8; D010088:Oxidoreductases; D011771:Pyruvate Oxidase; C040077:cytochrome bd terminal oxidase complex, E coli