When grown in aerated shaking culture, Bacillus subtilis expresses two different haem A-containing terminal oxidases: cytochrome aa3-quinol oxidase and cytochrome caa3 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 caa3 oxidase, and 67.6 kDa and 37.2 kDa for aa3-quinol oxidase. These features are in agreement with the sequence data for the corresponding structural genes in the aa3 and caa3 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.

Succinyl-CoA synthetase [succinate-CoA ligase (GDP-forming); EC 6.2.1.4] of rat liver, an a18 dimer, is a component of the enzymology of the tricarboxylic acid cycle and functions within the mitochondrial matrix. We have isolated and determined the sequence of a cDNA clone containing the coding sequence of the cytoplasmic precursor to the a subunit of this enzyme together with stretches of nontranslated sequence at the 5' and 3' ends. The translated amino acid sequence indicates the presence of a 27-residue N-terminal signal sequence for mitochondrial targeting. The amino acid sequence of the mature a subunit shows an extraordinary degree of homology to the a subunit of Escherichia coli succinyl-CoA synthetase, with >70% of the residues identical. This suggests that the fundamental differences in the quaternary structures and catalytic functions of the mammalian and bacterial enzymes must be attributable to differences in the .8 subunits. mRNA that hybridizes to the cloned DNA is 4800 nucleotide residues in length, confirming that each of the two subunits is encoded separately and does not arise by proteolysis of a primary gene product containing both subunits of the mature protein.