HIGH-YIELD PURIFICATION OF CYTOCHROME AA(3) AND CYTOCHROME CAA(3) OXIDASES FROM BACILLUS-SUBTILIS PLASMA-MEMBRANES

When grown in aerated shaking culture, Bacillus subtilis expresses two different haem A-containing terminal oxidases: cytochrome aa(3)-quinol oxidase and cytochrome caa(3) oxidase. This paper describes a high-yield conventional procedure for purifying the two haem A-containing oxidases from the same aerobic culture of Bacillus subtilis. Yields of close to 40% of the total haem A are achieved and about 6 mg of each of the purified oxidases is obtained from 4 litres of liquid culture. Both of the purified enzymes have two subunits, with apparent molecular masses of 71.6 kDa and 34.3 kDa for the cytochrome can, oxidase, and 67.6 kDa and 37.2 kDa for aa(3)-quinol oxidase. These features are in agreement with the sequence data for the corresponding structural genes in the aa(3) and caa(3) operons of B. subtilis. Some spectral and enzymic features of the two purified oxidases are reported that are consistent with the inclusion of both of these enzymes as members of the cytochrome oxidase superfamily.