D-lactate oxidation and generation of the proton electrochemical gradient in membrane vesicles from Escherichia coli GR19N and in proteoliposomes reconstituted with purified D-lactate dehydrogenase and cytochrome o oxidase

Biochemistry. 1986 May 6;25(9):2321-7. doi: 10.1021/bi00357a004.

Abstract

The respiratory chain in the cytochrome d deficient mutant Escherichia coli GR19N is a relatively simple, linear system consisting of primary dehydrogenases, ubiquinone 8, cytochrome b-556, and cytochrome o oxidase. By use of right-side-out and inside-out membrane vesicles from this strain, various oxidase activities and the generation of the H+ electrochemical gradient were studied. Oxidation of ubiquinol 1 or N,N,-N',N'-tetramethyl-p-phenylenediamine, which donate electrons directly to the terminal oxidase, generates a H+ electrochemical gradient comparable to that observed during D-lactate oxidation. In contrast, D-lactate/ubiquinone 1 or D-lactate/ferricyanide oxidoreductase activity does not appear to generate a membrane potential, suggesting that electron flow from D-lactate dehydrogenase to ubiquinone is not electrogenic. Moreover, proteoliposomes reconstituted with purified D-lactate dehydrogenase, ubiquinone 8, and purified cytochrome o catalyze D-lactate and ubiquinol 1 oxidation and generate a H+ electrochemical gradient similar to that observed in membrane vesicles. Strikingly, in inside-out vesicles, NADH oxidation generates a H+ electrochemical gradient that is very significantly greater than that produced by either D-lactate or ubiquinol 1; furthermore, NADH/ubiquinone 1 and NADH/ferricyanide oxidoreductase activities are electrogenic. It is suggested that the only component between D-lactate dehydrogenase or ubiquinol and oxygen in GR19N membranes that is directly involved in the generation of the H+ electrochemical gradient is cytochrome o, which functions as a "half-loop" (i.e., the oxidase catalyzes the scalar release of 2 H+ from ubiquinol on the outer surface of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

MeSH terms

  • Cell Membrane / metabolism
  • Electron Transport Complex IV / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Kinetics
  • L-Lactate Dehydrogenase / metabolism*
  • Lactates / metabolism*
  • Lactic Acid
  • Liposomes*
  • Mutation
  • Oxidation-Reduction
  • Proteolipids / metabolism*

Substances

  • Lactates
  • Liposomes
  • Proteolipids
  • proteoliposomes
  • Lactic Acid
  • L-Lactate Dehydrogenase
  • Electron Transport Complex IV