A NOVEL CYTOCHROME-C-OXIDASE FROM RHODOBACTER-SPHAEROIDES THAT LACKS CU-A

Rhodobacter sphaeroides contains at least two different cytochrome c o xidases. When these bacteria are grown with high aeration, the traditi onal aa(3)-type cytochrome c oxidase is present at relatively high lev els. However, under microaerophilic growth conditions or when the bact eria are grown photosynthetically, the amount of the aa(3)-type oxidas e is greatly diminished and an alternate cytochrome c oxidase is evide nt. This alternate oxidase has been purified and characterized. The en zyme consists of three subunits by SDS-PAGE analysis (M(app) 45, 35, a nd 29 kDa). Two of the three subunits (M(app) 35 and 29 kDa) contain c ovalently bound heme C. Metal and heme analyses indicate that the oxid ase contains heme C, heme B (protoheme IX), and Cu in a ratio of 3:2:1 . Cryogenic Fourier transform infrared (FTIR) difference spectroscopy of the CO adduct of the reduced enzyme shows that the oxidase contains a heme-copper binuclear center and, thus, is a member of the heme-cop per oxidase superfamily. In contrast to other members of this superfam ily, however, this oxidase does not contain either heme O or heme A as a component of the binuclear center, but has heme B at this site. The single equivalent of Cu found in the oxidase is accounted for by the CUB component at the binuclear center. This suggests that this oxidase does not contain Cu-A, which is found in all other well-characterized cytochrome c oxidases. Both EPR and optical spectroscopic studies are consistent with this conclusion, also indicating that this oxidase do es not contain Cu-A. Since the purified enzyme has a turnover number o f greater than 900 s(-1) using horse heart cytochrome c as a substrate , it is not likely that the lack of Cu-A is the result of damage incur red during the purification procedure. It is concluded that the altern ate cytochrome c oxidase is a novel cbb(3)-type of the heme-copper oxi dase superfamily that contains heme B at the binuclear center (not hem e O), and which lacks Cu-A.