Resonance Raman, infrared, and EPR investigation on the binuclear site structure of the heme-copper ubiquinol oxidases from Acetobacter aceti: effect of the heme peripheral formyl group substitution.

Acetobacter aceti produces two different terminal ubiquinol oxidases (cytochromes a1 and o) depending on the culture conditions. Two types of oxidases share a common protein moiety but with different heme components at the binuclear center (heme A for cytochrome a1 and heme O for cytochrome o). We investigated the structure of the binuclear site of the two oxidases using resonance Raman, Fourier transform-infrared (FT-IR), and EPR spectroscopies to clarify the interactions of heme A formyl group with protein moiety. We found that the overall architecture and the electronic configuration at the binuclear center in the oxidized state seem to be well conserved irrespective of the heme peripheral group at position 8, except for the azide-inhibited state. In contrast, we observed great variations in the C-N stretching frequency and cyanide-binding affinity in the CN-reduced state, in addition to multiple C-O stretching bands in the CO-reduced state. Present and previous studies suggest that the conformational flexibility of the binuclear center in the reduced ligand-bound state may be a common feature among the heme-copper oxidase superfamily. In the CN-reduced state, a hydrogen bond network may be formed among the formyl group, water molecule(s), and the surrounding amino acid residue(s). This network may be very important to maintain proper orientations of the distal amino acid residues and/or the CuB1+ ion relative to the cyanide ion bound to the ferrous heme iron and could play a critical role for the high affinity in cyanide binding.