Heme-linked ionization and chloride binding in intestinal peroxidase and lactoperoxidase

Abstract

Hog intestinal peroxidase and bovine lactoperoxidase exhibited similar spectral shifts upon pH alteration. From spectrophotometric titrations, it was found that there are hemelinked ionizations of p K a = 4.75 in intestinal peroxidase and pK a = 3.5 in lactoperoxidase. The apparent p K a (p K a′) increased with the increase in chloride concentration. The p K a′ vs log[Cl −] plots showed that the chloride forms complex with the acid forms of these enzymes with a dissociation constant (p K = 2.7). Although the dissociation constant ( K d) of the peroxidase-cyanide complexes is nearly independent of pH, cyanide competed with chloride in the acidic pH region. The slopes of log K d vs log[Cl −] were 1.0 for intestinal peroxidase and 0.5 for lactoperoxidase. The reaction of hydrogen peroxide with these peroxidases was also affected by chloride, similarly as the reaction with cyanide was. The results were explained by assuming that protonation occurs at the distal base and destroys the hydrogen bond between the base and a water molecule at the sixth coordinate position of the heme iron.