Characterization of the reaction between cytochrome c peroxidase (H52L) and potassium cyanide
The reactivity of potassium cyanide with a mutant of cytochrome c peroxidase in which the conserved distal histidine (His-52) was replaced with leucine [CcP(H52L)] was studied from pH 4 to 8 at 0.1 M ionic strength. Spectral studies were conducted to determine the coordination state of the heme in CcP(H52L) and in the CcP(H52L)/cyanide complex. Kinetic studies were performed to characterize the binding of cyanide to the enzyme as a function of pH. Based on the results of the study, it is suggested that HCN binds to the neutral pentacoordinate form of CcP(H52L). The association rate constant was found to be pH dependent while the dissociation rate constant was essentially independent of pH. The pH dependence of the association rate constant can be accounted for by a mechanism in which HCN can bind to either the neutral form or the hydroxy-ligated form of CcP(H52L). The iron-bound hydroxide assumes the role of base catalyst in CcP(H52L) just as His-52 does in native CcP. The dissociation rate constant for the CcP(H52L)/cyanide complex is about three times smaller than that for the native CcP/cyanide complex; this indicates slower dissociation of the ligand and suggests that His-52 may have a destabilizing effect on the bound cyanide by providing a proton to allow HCN to dissociate.