The Molecular Features and Catalytic Activity of Cu_A-Containing aco₃-Type Cytochrome c Oxidase from a Facultative Alkalophilic Bacillus

Abstract

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 Mrs of 50, 000, 41, 000, and 22, 000, and contains 1 molecule each of cytochrome α, cytochrome c, and cytochrome o₃, in the minimal structural unit (Mr, 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 αα₃-type cytochrome c oxidase. Resonance Raman studies demonstrated that the cytochrome a moiety was similar to that of an αα₃-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 520cm^-1, which is the same frequency as that of cytochrome αα₃•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 aco₃ except that the cytochrome aco₃ has cytochrome o₃ in place of cytochrome α₃ 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.