Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (Complex II)

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Abstract

Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium B subtilis consists of equimolar amounts of three polypeptides; a 65-kDa FAD-containing polypeptide, a 28-kDa iron-sulfur cluster containing polypeptide, and a 23-kDa membrane-spanning cytochrome b558 polypeptide, The enzyme complex was overproduced 2-3-fold in membranes of B. subtilis cells containing the sdhCAB operon on a low copy number plasmid and was purified in the presence of detergent. The cytochrome b558 subunit alone was similarly overexpressed in a complex II deficient mutant and partially purified. Isolated complex II catalyzed the reduction of various quinones and also quinol oxidation. Both activities were efficiently albeit not completely blocked by 2-n-heptyl-4-hydroxyquinoline N-oxide. Chemical analysis demonstrated two protoheme IX per complex II. One heme component was found to have an E(m,7.4) of +65 mV and an EPR g(max) signal at 3.68, to be fully reducible by succinate, and showed a symmetrical alpha-band absorption peak at 555 nm at 77 K. The other heme component was found to have an E(m,7.4) of -95 mV and an EPR g(max) signal at 3.42, was not reducible by succinate under steady-state conditions, and showed in the reduced state an apparent split alpha-band absorption peak with maxima at 553 and 558 nm at 77 K. Potentiometric titrations of partially purified cytochrome b558 subunit demonstrated that the isolated cytochrome b558 also contains two hemes. Some of the properties, i.e., the alpha-band light absorption peak at 77 K, the line shapes of the EPR g(max) signals, and reactivity with carbon monoxide were observed to be different in B. subtilis cytochrome b558 isolated and in complex II. This suggests that the bound flavoprotein and iron-sulfur protein subunits protect or affect the heme environment in the assembled complex

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External organisations
  • Lund University
  • Chalmers University of Technology
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Microbiology
  • Biochemistry and Molecular Biology
Original languageEnglish
Pages (from-to)7411-7421
JournalBiochemistry
Volume31
Publication statusPublished - 1992
Publication categoryResearch
Peer-reviewedYes