PURIFICATION AND CHARACTERIZATION OF THE SUCCINATE-DEHYDROGENASE COMPLEX AND CO-REACTIVE B-TYPE CYTOCHROMES FROM THE FACULTATIVE ALKALIPHILE BACILLUS-FIRMUS OF4

The presence of a cytochrome be-type terminal oxidase in Bacillus firm us OF4 had been suggested from the effects of CO on the spectra of red uced membrane cytochromes (Hicks, D.B., Plass, R.J. and Quirk, P.G. (1 991) J. Bacteriol. 173, 5010-5016). In that study the GO-binding b-typ e cytochrome was partially purified by anion exchange chromatography. No further purification was attempted but later HPLC analysis indicate d the absence of significant heme O in the B. firmus OF4 membranes. Th e current work shows that the partially purified cytochrome b is actua lly composed of three different b-type cytochromes which can be separa ted and purified by a combination of ion-exchange, hydroxyapatite and gel filtration chromatographies. Two of the cytochromes were GO-reacti ve but lacked the characteristic multisubunit composition of known ter minal oxidases. Neither purified cytochrome catalyzed quinol or ferroc ytochrome c oxidation. The more abundant GO-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 pu rified and showed two bands of 23 and 17 kDa on SDS-PAGE. The function s of the cytochromes b(560) and b(558) remain unknown but together the y account for the spectrum originally attributed to cytochrome be. The third, non-CO reactive, cytochrome b was associated with substantial succinate dehydrogenase activity and was purified as a three subunit s uccinate dehydrogenase complex with high specific activity (17.7 mu mo l/min/mg). Limited N-terminal sequence of each subunit demonstrated ma rked similarity to the complex from Bacillus subtilis. The cytochrome b of the alkaliphile enzyme was reduced about 50% by succinate compare d to the level of reduction achieved by dithionite. The enzyme reacted with both napthoquinones and benzoquinones. The results presented ind icate that Bacillus firmus OF4 contains a succinate dehydrogenase comp lex with very similar properties to the enzyme from Bacillus subtilis, but does not contain a cytochrome o-type terminal oxidase under the g rowth conditions studied.