Effects of 2,4-substituents of deuteropheme upon redox potentials of horseradish peroxidases.

The effects of pH on redox potentials of horseradish peroxidase-A and -(B + C) and of their heme-substituted enzymes with mesoheme, deuteroheme, chlorocruoroheme, and diacetyldeuteroheme were investigated. The slope in the plot of E_o against pK₃ (a measure of basicity of pyrrole nitrogen) was found to be close upon 59 $\text{mV}\text{p}\text{K}{}_3$ unit. It was also found that the ratio of ΔpK_r to ΔpK₃ was about 0.1 while that of ΔpK_o to ΔpK₃ was almost unity. Here, K_r and K_o stand for heme-linked proton dissociation constants in the ferrous and ferric peroxidases, respectively. The difference of either pK_r or pK_o between two isoenzyme preparations was about 1.6. These results support the previous conclusion (Arch. Biochem. Biophys. 165, 725, 1974 that K_r represents a proton dissociation constant of a distal amino acid residue and that there is a strong hydrogen bonding between its base and the water oxygen atom as a sixth ligand in the ferric state of peroxidases. The difference of redox potentials at pH 8.5 between two natural isoenzyme preparations, amounting to 88 mV, was attributed to the change in the hydrogen bonding strength caused by the difference in basicity of two distal amino acid residues. A possibility that approximate redox potentials of hemoproteins can be determined by analysis of several factors is discussed.