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

Biochemistry. 1992 Aug 18;31(32):7411-21. doi: 10.1021/bi00147a028.


Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium Bacillus 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 Em,7.4 of +65 mV and an EPR gmax 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 Em,7.4 of -95 mV and an EPR gmax 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 gmax 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / enzymology*
  • Bacillus subtilis / genetics
  • Cell Membrane / enzymology
  • Cytochrome b Group / chemistry
  • Cytochrome b Group / isolation & purification
  • Cytochrome b Group / metabolism
  • Electron Spin Resonance Spectroscopy
  • Electron Transport Complex II
  • Escherichia coli / genetics
  • Flavin-Adenine Dinucleotide / analysis
  • Heme / analysis*
  • Kinetics
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / isolation & purification
  • Multienzyme Complexes / metabolism
  • NADPH Oxidases*
  • Operon
  • Oxidoreductases / chemistry*
  • Oxidoreductases / isolation & purification
  • Oxidoreductases / metabolism
  • Potentiometry
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Spectrophotometry
  • Succinate Dehydrogenase / chemistry*
  • Succinate Dehydrogenase / isolation & purification
  • Succinate Dehydrogenase / metabolism


  • Cytochrome b Group
  • Multienzyme Complexes
  • Recombinant Proteins
  • Flavin-Adenine Dinucleotide
  • Heme
  • cytochrome b558
  • Oxidoreductases
  • Electron Transport Complex II
  • Succinate Dehydrogenase
  • NADPH Oxidases