MOLECULAR-GENETIC AND PROTEIN CHEMICAL CHARACTERIZATION OF THE CYTOCHROME BA(3) FROM THERMUS-THERMOPHILUS HB8

Thermus thermophilus HB8 cells grown under reduced dioxygen tensions c ontain a substantially increased amount of heme A, much of which appea rs to be due to the presence of the terminal oxidase, cytochrome ba,. We describe a purification procedure for this enzyme that yields simil ar to 100 mg of pure protein from 2 kg of wet mass of cells grown in l ess than or equal to 50 mu M O-2. Examination of the protein by SDS-po lyacrylamide gel electrophoresis followed by staining with Coomassie B lue reveals one strongly staining band at similar to 35 kDa and one ve ry weakly staining band at similar to 18 kDa as reported earlier (Zimm ermann, B. H., Nitsche, C. I., Fee, J. A., Rusnak, F., and Munck, E. ( 1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5779-5783). By contrast, tre atment of the gels with AgNO3 reveals that the larger polypeptide stai ns quite weakly while the smaller polypeptide stains very strongly. Th ese results suggested the presence of two polypeptides in this protein . Using partial amino acid sequences from both proteins to obtain DNA sequence information, we isolated and sequenced a portion of the Therm us chromosome containing the genes encoding the larger protein, subuni t I (cbaA), and the smaller protein, subunit II (cbaB). The two polype ptides were isolated using reversed phase liquid chromatography, and t heir mole percent amino acid compositions are consistent with the prop osed translation of their respective genes. The two genes appear to be part of a larger operon, but we have not extended the sequencing to i dentify initiation and termination sequences. The deduced amino acid s equence of subunit I includes the six canonical histidine residues inv olved in binding the low spin heme B and the binuclear center Cu-B/hem e A. These and other conserved amino acids are placed along the polype ptide among alternating hydrophobic and hydrophilic segments in a patt ern that shows clear homology to other members of the heme- and copper -requiring terminal oxidases. The deduced amino acid sequence of the s ubunit II contains the Cu-A binding motif, including two cysteines, tw o histidines, and a methionine, but, in contrast to most other subunit s II, it has only one region of hydrophobic sequence near its N termin us. Alignment of these two polypeptides with other cytochrome c and qu inol oxidases, combined with secondary structure analysis and previous spectral studies, clearly establish cytochrome ba(3) as a bona fide m ember of the superfamily of heme- and copper-requiring oxidases. The a lignments further indicate that cytochrome ba(3) is phylogenetically d istant from other cytochrome c and quinol oxidases, and they substanti ally decrease the number of conserved amino acid residues.