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 ac tivity of horseradish peroxidase (HRP), we have expressed the R38A, R3 8H, and R38H/H42V mutants. The R38A HRP mutant gives a normal compound I species with H2O2 that is reduced by ferrocyanide to the ferric sta te without the detectable formation of a compound II species. In the c ase of the R38H and R38H/H42V mutants, compound I itself is only detec ted by stopped flow methods. The rates of compound I formation at 4 de grees C are 8.0 x 10(4), 1.3 x 10(6), and 1.6 x 10(3) M-1 s(-1) for th e 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 3 2-fold more slowly than the wild-type enzyme. The apparent k(cat)/K-m values for thioanisole sulfoxidation and styrene epoxidation indicate that the reaction efficiencies of the R38H and wild-type enzymes are c omparable. However, the R38A and R38H/H42V mutants are 190- and 1400-f old more efficient as sulfoxidation catalysts, and 25- and 26-fold mor e efficient as styrene epoxidation catalysts, respectively, than the w ild-type enzyme. Thus, even though Arg38 plays a role in the formation and stabilization of compounds I and II, its replacement by other res idues can be used to improve peroxygenative catalysis.