RESCUE OF THE CATALYTIC ACTIVITY OF AN H42A MUTANT OF HORSERADISH-PEROXIDASE BY EXOGENOUS IMIDAZOLES

His-42 plays a critical role in the H2O2-dependent catalytic turnover of horseradish peroxidase (HRP), This is clearly illustrated by the fi nding that an H42A mutation decreases the rate of Compound I formation by a factor of similar to 10(6). As shown here, the addition of 2-sub stituted imidazoles partially rescues both the rate of formation of Co mpound I and the peroxidase activity of the H42A mutant, a-Substituted imidazoles are the most effective because they do not coordinate to t he iron, In contrast to native HRP, which exhibits a parabolic pH prof ile, and the H42A mutant, for which the activity increases linearly wi th increasing pH, the activity of the H42A mutant in the presence of 1 ,2-dimethylimidazole (pK(a) = 8.0) exhibits a sigmoidal pH dependence with a midpoint at pH 8.0 +/- 0.2. Similar results are obtained with 2 -methylimidazole. These results establish that the free base forms of these imidazoles facilitate HRP turnover, The spectroscopic binding co nstants for 1,2-dimethylimidazole and 2-methylimidazole are K-d = 2.9 +/- 1.3 and 2.5 +/- 0.2 M, respectively. When cyanide is bound to the heme, the K-d for 1,2-dimethylimidazole is 0.17 M. This > 10-fold decr ease in K-d may reflect hydrogen bonding of the protonated imidazole t o the iron-coordinated cyanide, The log of the rate of Compound I form ation exhibits a linear dependence on the molecular volume of the imid azoles used to rescue the activity, If the rates are corrected for dif ferences in the size of the imidazoles, the log of the rates is linear ly related to the pK(a) of the imidazoles. This Bronsted analysis pred icts that similar to 60% of a positive charge develops on the imidazol e in the transition state of Compound I formation, The results confirm the acid-base role of the distal histidine, demonstrate that exogenou s histidines can function as surrogates for the missing histidine in t he H42A mutant, and provide a transition state model of relevance to t he formation of Compound I in the native protein.