Reconstitution of the Ubiquinone-dependent pyruvate oxidase system of Escherichia coli with the cytochrome o terminal oxidase complex.
The aerobic respiratory chain of Escherichia coli is branched and contains two terminal oxidases. The chain predominant when the cells are grown with low aeration terminates with the cytochrome d terminal oxidase complex, and the branch present under high aeration ends with the cytochrome o terminal oxidase complex. Previous work has shown that cytochrome d complex functions as a ubiquinol-8 oxidase, and that a minimal respiratory chain can be reconstituted in proteoliposomes with a flavoprotein dehydrogenase (pyruvate oxidase), ubiquinone-8, and the cytochrome d complex. This paper demonstrates that the cytochrome o complex functions as an efficient ubiquinol-8 oxidase in reconstituted proteoliposomes, and that ubiquinone-8 serves as an electron carrier from the flavoprotein to the cytochrome complex. The maximal turnover (per cytochrome o) achieved in reconstituted proteoliposomes is at least as fast as observed in E. coli membrane preparations. Electron flow from the flavoprotein to oxygen in the reconstituted proteoliposomes generates a transmembrane potential of at least 120 mV, negative inside, which is sensitive to ionophore uncouplers and inhibitors of the terminal oxidase. These data demonstrate the minimal composition of this respiratory chain as a flavoprotein dehydrogenase, ubiquinone-8, and the cytochrome o complex. Previous models have suggested that cytochrome b556, also a component of the E. coli inner membrane, is required for electron flow to cytochrome o. This is apparently not the case. It now is clear that both of the E. coli terminal oxidases act as ubiquinol-8 oxidases and, thus, ubiquinone-8 is the branch point between the two respiratory chains.
D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group; D003580:Cytochromes / Q000302:isolation & purification; D003576:Electron Transport Complex IV; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D029968:Escherichia coli Proteins; D007700:Kinetics; D008954:Models, Biological; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010101:Oxygen Consumption; D011771:Pyruvate Oxidase / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014451:Ubiquinone / Q000378:metabolism*
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D014451:Ubiquinone; C017168:cytochrome bo, E coli; C030778:ubiquinone 8; D010088:Oxidoreductases; D011771:Pyruvate Oxidase; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV