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 (Mapp 45, 35, and 29 kDa). Two of the three subunits (Mapp 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 CuB component at the binuclear center. This suggests that this oxidase does not contain CuA, 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 CuA. 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 CuA 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 CuA.
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