Abstract
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.
Highlights
The cytochrome d complex from Escherichia coli has oratory aerobicgrowth conditions either oxidasealone is been reconstituted in proteoliposomes
The production of a A+ by the cytochrome d complex in the vesicles and theeffects of CCCP and nigericin suggested that a proton gradient is formed concomitant with quinol oxidase activity
As noted before (4), this dyewillrespond to a A$ formed by a K+-diffusion potential in thepresence of valinomycin at low lipid concentrations and good agreement has been reported between AI) values determined by this method and by flowdialysis (7,8)
Summary
Preparation of Proteoliposomes-Formation of artificial liposomes containing the cytochrome d complex in the bilayer was done by the detergent dialysis method originally described by Racker and coworkers (for a review see Ref. 12). The assay solution consisted of 0.1p M 9-amino acridine and vesicles contained 100 pg of phospholipid and 1pg of the cytochrome d complex in 3 ml of 50 mM potassium phosphate, 2 mM DTE, 1 mM EDTA, pH 7.5. The assay was performed in a solution consisting of vesicles containing 500 pgof phospholipid and 5 pgof the cytochrome d complex in 3 ml of 1 mM EDTA, 2 mM DTE, 50 mM KCl, pH 7.0. The reaction mixture consisted of vesicles containing 300 pg of cytochrome d complex (6 nmol of protoheme IX) and 30 mg of phospholipid in a buffer consisting of 0.1 mM EDTA, 2 p~ valinomycin, 2 mM DTE, 150 mM KCl, 50 mM sucrose, pH 7.5. A YSI model 53 oxygen electrode was used.The rateof 0 2 consumption was compared to that of proteoliposomes that had been incubated with control antibodies that did not bindto the cytochrome
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