The cytochrome d complex is a coupling site in the aerobic respiratory chain of Escherichia coli.
The cytochrome d complex from Escherichia coli has been reconstituted in proteoliposomes. Previous studies have shown that the enzyme rapidly oxidizes ubiquinol-8 within the bilayer as well as the soluble homologue, ubiquinol-1, and that quinol oxidase activity is accompanied by the formation of a transmembrane potential across the vesicle bilayer. In this work, the proton pumping activity of the cytochrome in the reconstituted vesicles is examined. Ubiquinol-1 oxidase activity is shown to be accompanied by the net alkalinization of the interior space of the reconstituted vesicles and by the release of protons in the external volume. H+/O ratios varying from 0.6 to 1.2 were measured in different preparations, by the oxygen pulse technique. Antibodies which bind specifically to subunit I (cytochrome b558) of the 2-subunit oxidase were used to estimate the topology of the reconstituted oxidase in the vesicles. It was concluded that 70-85% of the molecules were oriented with subunit I facing the outside and that this population of molecules is responsible for the observed proton release. Correction for the fraction of the oxidase which pumps protons into the vesicle interior yields an estimate of H+/O = 1.7 +/- 0.2. It is proposed that the enzyme does not function as an actual proton pump, but that the enzyme oxidizes ubiquinol and reduces oxygen (to water) on opposite faces of the membrane. Hence, scalar chemistry would yield H+/O = 2 and an electrogenic reaction by virtue of the transmembrane electron transfer between the proposed active sites.
M J
MJ
Miller
R B
RB
Gennis
D000332:Aerobiosis; D000911:Antibodies, Monoclonal; D052160:Benzothiazoles; D002232:Carbocyanines; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D017937:Cytochrome d Group; D003580:Cytochromes / Q000378:metabolism*; D004579:Electron Transport; D004926:Escherichia coli / Q000378:metabolism*; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007158:Immunologic Techniques; D007473:Ion Channels / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D010101:Oxygen Consumption; D011522:Protons; D011808:Quinone Reductases / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D052160:Benzothiazoles; D002232:Carbocyanines; D003580:Cytochromes; D005456:Fluorescent Dyes; D007473:Ion Channels; D008081:Liposomes; D011522:Protons; C012944:3,3'-dipropyl-2,2'-thiadicarbocyanine; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D017937:Cytochrome d Group; D011808:Quinone Reductases; C030914:ubiquinol-1 oxidase