Modified, Large-Scale Purification of the Cytochrome O Complex (Bo-Type Oxidase) of Escherichia Coli Yields a Two heme/one Copper Terminal Oxidase With High Specific Activity

Biochemistry. 1992 Aug 4;31(30):6917-24. doi: 10.1021/bi00145a008.

Abstract

The cytochrome o complex is a bo-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. This complex has a close structural and functional relationship with the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. The specific activity, subunit composition, and metal content of the purified cytochrome o complex are not consistent for different preparative protocols reported in the literature. This paper presents a relatively simple preparation of the enzyme starting with a strain of Escherichia coli which overproduces the oxidase. The pure enzyme contains four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Partial amino acid sequence data confirm the identities of subunit I, II, and III from the SDS-PAGE analysis as the cyoB, cyoA, and cyoC gene products, respectively. A slight modification of the purification protocol yields an oxidase preparation that contains a possible fifth subunit which may be the cyoE gene product. The pure four-subunit enzyme contains 2 equivs of iron but only 1 equiv of copper. There is no electron paramagnetic resonance detectable copper in the purified enzyme. Hence, the equivalent of CuA of the aa3-type cytochrome c oxidases is absent in this quinol oxidase. There is also no zinc in the purified quinol oxidase. Finally, monoclonal antibodies are reported that interact with subunit II. One of these monoclonals inhibits the quinol oxidase activity of the detergent-solubilized, purified oxidase. Hence, although subunit II does not contain CuA and does not interact with cytochrome c, it still must have an important function in the bo-type ubiquinol oxidase.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Ascorbic Acid / pharmacology
  • Copper / analysis*
  • Electron Spin Resonance Spectroscopy
  • Electron Transport Complex IV / chemistry
  • Electron Transport Complex IV / genetics
  • Electron Transport Complex IV / isolation & purification*
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / enzymology*
  • Ethylenediamines / pharmacology
  • Heme / analysis*
  • Molecular Sequence Data
  • Operon
  • Peptide Fragments / chemistry

Substances

  • Ethylenediamines
  • Peptide Fragments
  • N,N,N',N'-tetramethylethylenediamine
  • Heme
  • Copper
  • cytochrome o oxidase
  • Electron Transport Complex IV
  • Ascorbic Acid