Purification and characterization of the succinate dehydrogenase complex and CO-reactive b-type cytochromes from the facultative alkaliphile Bacillus firmus OF4

Abstract

The presence of a bo-type terminal oxidase in Bacillus firmus OF4 had been suggested from the effects of CO on the spectra of reduced membrane cytochromes (Hicks, D.B., Plass, R.J. and Quirk, P.G. (1991) J. Bacteriol. 173, 5010–5016). In that study the CO-binding b-type cytochrome was partially purified by anion exchange chromatography. No further purification was attempted but later HPLC analysis indicated the absence of significant heme O in the B. firmus OF4 membranes. The current work shows that the partially purified cytochrome b is actually composed of three different b-type cytochromes which can be separated and purified by a combination of ion-exchange, hydroxyapatite and gel filtration chromatographies. Two of the cytochromes were CO-reactive but lacked the characteristic multisubunit composition of known terminal oxidases. Neither purified cytochrome catalyzed quinol or ferrocytochrome c oxidation. The more abundant CO-reactive b-type cytochrome (cytochrome b 560) had an apparent molecular mass of 10 kDa, whereas the other, more minor component (cytochrome b 558), was partially purified and showed two bands of 23 and 17 kDa on SDS-PAGE. The functions of the cytochromes b 560 and b 558 remain unknown but together they account for the spectrum originally attributed to cytochrome bo. The third, non-CO reactive, cytochrome b, was associated with substantial succinate dehydrogenase activity and was purified as a three subunit succinate dehydrogenase complex with high specific activity (17.7 μmol/min/mg). Limited N-terminal sequence of each subunit demonstrated marked similarity to the complex from Bacillus subtilis. The cytochrome b of the alkaliphile enzyme was reduced about 50% by succinate compared to the level of reduction achieved by dithionite. The enzyme reacted with both napthoquinones and benzoquinones. The results presented indicate that Bacillus firmus OF4 contains a succinate dehydrogenase complex with very similar properties to the enzyme from Bacillus subtilis, but does not contain a cytochrome o-type terminal oxidase under the growth conditions studied.