Improvement of peroxygenase activity by relocation of a catalytic histidine within the active site of horseradish peroxidase.

To examine the role of Arg38 in the peroxidative and peroxygenative activity of horseradish peroxidase (HRP), we have expressed the R38A, R38H, and R38H/H42V mutants. The R38A HRP mutant gives a normal compound I species with H2O2 that is reduced by ferrocyanide to the ferric state without the detectable formation of a compound II species. In the case of the R38H and R38H/H42V mutants, compound I itself is only detected by stopped flow methods. The rates of compound I formation at 4 degrees C are 8.0 x 10(4), 1.3 x 10(6), and 1.6 x 10(3) M-1 s-1 for the R38A, R38H, and R38H/H42V mutants, respectively. The R38A, R38H, and R38H/H42V mutants oxidize guaiacol 10-, 2-, and 55-fold, respectively, more slowly than the wild-type enzyme and oxidize ABTS 6-, 3-, and 32-fold more slowly than the wild-type enzyme. The apparent kcat/K(m) values for thioanisole sulfoxidation and styrene epoxidation indicate that the reaction efficiencies of the R38H and wild-type enzymes are comparable. However, the R38A and R38H/H42V mutants are 190- and 1400-fold more efficient as sulfoxidation catalysts, and 25- and 26-fold more efficient as styrene epoxidation catalysts, respectively, than the wild-type enzyme. Thus, even though Arg38 plays a role in the formation and stabilization of compounds I and II, its replacement by other residues can be used to improve peroxygenative catalysis.