EFFECTS OF IMIDAZOLES AND PH ON THE PEROXIDASE-ACTIVITY OF THE HEMIN HYDROGEN-PEROXIDE SYSTEM

The reaction of hydrogen peroxide with protohemin has been examined in aqueous solution (pH 5-10) with potassium ferrocyanide as a substrate . Kinetic studies were carried out in the presence and absence of imid azole bases(B: imidazole, 1-methylimidazole, 2-methylimidazole, 4-meth ylimidazole and 1,2-dimethylimidazole). The apparent second-order rate constant (k(app)) for the formation of ferricyanide was used as the i ndex of the peroxidase activity. in the absence of bases, hemin showed weak and constant activity at alkaline pH but has lost it at acidic p H. A plot of log k(app) vs pH has revealed that the association of one H+ equivalent with apparent pK(a) = 6.9 has inhibited the reaction. A long with the pH-dependency of the absorption spectra of ferric hemin, it was suggested that the hydroxide coordination to the ferric hemin is essential to the generation of the peroxidase activity. On the othe r hand, addition of bases resulted in an enhancement of the reaction r ate at alkaline pH. This effect was well. accounted for by the mono-li gation of a base to the iron atom in the high-valent porphyrin interme diate(s). However, the reaction was found to be inhibited in the prese nce of large excess of unhindered bases, and the inhibition was strong ly correlated to the formation of bis-coordinated ferric hemin. Theref ore, bis-ligating bases have prevented the access of H2O2 to the ferri c hemin and the subsequent formation of the intermediates. At acidic p H, the bis-coordination was promoted and the unhindered bases were les s effective activators. Addition of sterically hindered 2-methylimidaz ole, by contrast, resulted in the higher activity at acidic pH than th at in the base-free system. This may suggest that oxo-ligand in the in termediate was activated by the hydrogen bonding of the protonated 2-m ethylimidazole. A reaction scheme that satisfies the above criteria is proposed, and the correlation with the reactions of heme enzymes is d iscussed.