KINETICS OF CYANIDE AND FLUORIDE BINDING BY FERRIC HORSERADISH PEROXIDASE

The kinetics of the reversible binding of cyanide by ferric horseradish peroxidase has been studied over the pH range 4.2-11.3 by means of a stopped-flow apparatus. Analysis of the pH dependence of the bimo-lecular rate constants is consistent with the presence of three heme-linked ionizable groups on peroxidase with pK values of 4.1, 6.4, and 10.8. It is not possible to distinguish whether HCN or CN- is the attacking species, but the bound form of the ligand appears to be the cyanide ion. The kinetic results obtained for fluoride binding by horseradish peroxidase are reanalyzed, and it is shown that the published mechanism requires extension because it violates the principle of detailed balancing. A satisfactory mechanism for the pH range 4.1-7.9 postulates that fluoride ion binds to peroxidase which contains two heme-linked acid groups with pK values of 4.3 and 6.1. These are in agreement with the results of the cyanide kinetic analysis. © 1968, American Chemical Society. All rights reserved.