The molecular features and catalytic activity of Cu_a-containing aco₃ -type cytochrome c oxidase from a facultative alkalophilic Bacillus

Cytochrome aco₃ of Bacillus YN-2000 was purified by an improved procedure and its molecular features and catalytic activity were extensively studied. The enzyme molecule was composed of three subunits with M_rs of 50, 000, 41, 000, and 22, 000, and contains 1 molecule each of cytochrome a, cytochrome c, and cytochrome o₃ in the minimal structural unit (M_r, 113, 000). The 41, 000 subunit (subunit II) contains heme c. The EPR, optical, and resonance Raman spectra of the oxidized enzyme demonstrated the presence of Cu_A whose coordination environment bore close resemblance to that of the aa₃ type cytochrome c oxidase. Resonance Raman studies demonstrated that the cytochrome a moiety was similar to that of an aa₃-type oxidase and also that the cytochrome o₃ contained a five-coordinated high-spin heme with histidine as an axial ligand. The Fe-CO stretching mode of the cytochrome o₃*CO complex was observed at 520 cm^-1, which is the same frequency as that of cytochrome aa₃* CO. The oxygen consumption activity of cytochrome aco₃ was measured using several kinds of cytochromes c as the electron mediators. The reaction between cytochrome aco₃ and eucaryotic cytochromes c was completely inhibited by poly-L-lysine. In contrast, poly-L-lysine was indispensable for sufficient reaction between the oxidase and Bacillus YN-2000 cytochrome c-553, the physiological electron donor. The combined results on the structure and enzymatic properties suggest that the cytochrome aco₃ is very similar to cytochrome caa₃ except that the cytochrome aco₃ has cytochrome 0₃ in place of cytochrome 0₃ and the cytochrome c component has a very low redox midpoint potential (95 mV). A possible relationship between the role of the low redox potential cytochrome c and the bioenergetic properties of the alkalophilic bacterium is discussed.