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The proximal iron ligand in horseradish peroxidase (HRP) is His-170. The H170A mutant of polyhistidine-tagged HRP (hHRP) has been expressed in a baculovirus system and has been purified and characterized. At pH 7, the Soret maximum of the mutant is at 414 nm rather than 403 nm. Resonance Raman spectra indicate that the protein is primarily 6-coordinate low-spin in the ferric state with a band in the ferrous state at 212 cm-1 indicative of distal histidine coordination to the iron. Exogenous imidazole (Im) binds to the enzyme with Kd = 22 +/- 4 mM. Reaction of H170A hHRP with H2O2 does not give spectroscopically detectable compound I or compound II intermediates but results in gradual degradation of the heme group. Nevertheless, H170A hHRP is catalytically active, and its guaiacol and ABTS peroxidase activities are improved 260- and 125-fold, respectively, in the presence of saturating concentrations of Im. The Km for the stimulatory effect of Im is 24 mM for both guaiacol and ABTS. The pH profile of H170A hHRP differs from that of wild-type hHRP, but the differences are essentially eliminated by Im. The rate of formation of "compound I" for H170A hHRP, determined by steady state kinetic methods, is k1 = 16 M-1 s-1 without Im and k1 = 2.4 x 10(4) M-1 s-1 with Im. The corresponding rate for wild-type hHRP is k1 = 4.4 x 10(6) M-1 s-1. The results indicate that Im binds in the cavity created by the H170A mutation, coordinates to the heme iron atom, and restores a large part of the catalytic activity by rescuing the rate of compound I formation. However, this rescue of the catalytic activity by Im is possibly limited by coordination of the heme to the distal histidine (His-42) in the H170A mutant. Thus, a primary function of the proximal histidine is to tether the iron atom to disfavor sixth ligand binding, particularly coordination of the iron to the distal histidine. In addition, strong hydrogen bonding of the proximal ligand may be critical for facilitating O-O bond cleavage in the formation of compound I.

His-42 plays a critical role in the H2O2-dependent catalytic turnover of horseradish peroxidase (HRP). This is clearly illustrated by the finding that an H42A mutation decreases the rate of Compound I formation by a factor of approximately10(6). As shown here, the addition of 2-substituted imidazoles partially rescues both the rate of formation of Compound I and the peroxidase activity of the H42A mutant. 2-Substituted imidazoles are the most effective because they do not coordinate to the iron. In contrast to native HRP, which exhibits a parabolic pH profile, and the H42A mutant, for which the activity increases linearly with increasing pH, the activity of the H42A mutant in the presence of 1,2-dimethylimidazole (pKa = 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 constants for 1, 2-dimethylimidazole and 2-methylimidazole are Kd = 2.9 +/- 1.3 and 2. 5 +/- 0.2 M, respectively. When cyanide is bound to the heme, the Kd for 1,2-dimethylimidazole is 0.17 M. This >10-fold decrease in Kd may reflect hydrogen bonding of the protonated imidazole to the iron-coordinated cyanide. The log of the rate of Compound I formation exhibits a linear dependence on the molecular volume of the imidazoles used to rescue the activity. If the rates are corrected for differences in the size of the imidazoles, the log of the rates is linearly related to the pKa of the imidazoles. This Brönsted analysis predicts that approximately 60% of a positive charge develops on the imidazole in the transition state of Compound I formation. The results confirm the acid-base role of the distal histidine, demonstrate that exogenous histidines can function as surrogates for the missing histidine in the H42A mutant, and provide a transition state model of relevance to the formation of Compound I in the native protein.