A Novel Cytochrome c Oxidase from Rhodobacter sphaeroides That Lacks Cu_A

Rhodobacter sphaeroides contains at least two different cytochrome c oxidases. When these bacteria are grown with high aeration, the traditional aa₃-type cytochrome c oxidase is present at relatively high levels. However, under microaerophilic growth conditions or when the bacteria are grown photosynthetically, the amount of the aa₃-type oxidase is greatly diminished and an alternate cytochrome c oxidase is evident. This alternate oxidase has been purified and characterized. The enzyme consists of three subunits by SDS—PAGE analysis (M_app 45, 35, and 29 kDa). Two of the three subunits (M_app 35 and 29 kDa) contain covalently bound heme C. Metal and heme analyses indicate that the oxidase 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–copper oxidase superfamily. In contrast to other members of this superfamily, 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 Cu_B 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 does not contain Cu_A. Since the purified enzyme has a turnover number of 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 incurred during the purification procedure. It is concluded that the alternate cytochrome c oxidase is a novel cbb₃-type of the heme-copper oxidase superfamily that contains heme B at the binuclear center (not heme O), and which lacks Cu_A.