Purification and Reconstitution of the Cytochrome d Terminal Oxidase Complex from Escherichia coli

Michael J. Miller, Robert B. Gennis

Research output: Contribution to journalArticlepeer-review

Abstract

This chapter describes the purification of the cytochrome d complex and the incorporation of the complex into artificial proteoliposomes. Escherichia coli is capable of producing two different terminal oxidases, the cytochrome o and cytochrome d complexes. The cytochrome d complex is produced when oxygen is limited and the cytochrome o complex predominates when high concentrations of oxygen are present. Formation of artificial proteoliposomes containing the cytochrome d complex is accomplished by the detergent dialysis method. Cytochrome d incorporated in the proteoliposomes generate a proton gradient across the membrane with ubiquinol-1 as a substrate. The quenching of the fluorescence of carbocyanine dyes can be used to measure the electrical potential gradient formed concomitant with quinol oxidase activity. Pyruvate oxidase is a flavoprotein dehydrogenase found in E. coli that, although it couples to the aerobic respiratory chain, can be isolated in a water-soluble form. When the flavin prosthetic group is reduced by the addition of the substrate pyruvate, the enzyme acquires an enhanced affinity for lipids and membranes. This enzyme functions as a ubiquinone-8 reductase in vitro and in vivo.

Original languageEnglish (US)
Pages (from-to)87-94
Number of pages8
JournalMethods in enzymology
Volume126
Issue numberC
DOIs
StatePublished - Jan 1 1986

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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