Horseradish peroxidase. Complex formation with anions and hydrocyanic acid.

T Araiso,H.B Dunford

doi:10.1016/s0021-9258(19)68748-x

Abstract

Equilibrium binding experiments have been performed with perchlorate, chloride, and acetate in the presence of horseradish peroxidase. The binding of perchlorate and acetate appears to be like that of nitrate, at a site other than the sixth coordination position of the heme iron. Competitive experiments using both nitrate and cyanide demonstrate that two different binding sites are present on the enzyme. Chloride appears to bind at the sixth coordination position as do both fluoride and cyanide. Temperature jump experiments indicate that it is likely the nitrate anion and not undissociated nitric acid which is the binding species. Competitive stopped flow experiments indicate that the bound nitrate slows both the association rate and dissociation rate of cyanide, indicating that nitrate binds close to the sixth coordination position.

Highlights

Equilibriumbindingexperimentshavebeenperformed with perchlorate, chloride, and acetate in the presence of horseradishperoxidase
The bindingof perchlorate and acetate appears to be like that of nitrate, at a site other than the sixth coordination position of the heme iron. Competitive experiments using both nitrate and cyanide demonstrate that wo different binding sites are present on the enzyme
Chloride where M o b s is the absorbance change observed upon anion binding

Summary

COMPLEX FORMATION WITH ANIONS AND HYDROCYANIC ACID*

The bindingof perchlorate and acetate appears to be like that of nitrate, at a site other than the sixth coordination position of the heme iron. Competitive experiments using both nitrate and cyanide demonstrate that wo different binding sites are present on the enzyme. In abrief communication the binding of nitrate tohorseradish peroxidase at low pH and its influence on compound I formation have been described (1). It was tacitly assumed, without proof, that nitrate and not nitric acid is the binding species and that only protonated enzyme reacts with nitrate as shown below: E

KaE It H’

RESULTS

Dissociation constants for complexes of horseradish peroxidase

KGE EH'

Reverse rate constants