Ionic strength and pH effect on the Fe(III)-imidazolate bond in the heme pocket of horseradish peroxidase: an EPR and UV–visible combined approach

Abstract

The effects of chloride, dihydrogenphosphate and ionic strength on the spectroscopic properties of horseradish peroxidase in aqueous solution at pH=3.0 were investigated. A red-shift ( λ=408 nm) of the Soret band was observed in the presence of 40 mM chloride; 500 mM dihydrogenphosphate or chloride brought about a blue shift of the same band ( λ=370 nm). The EPR spectrum of the native enzyme at pH 3.0 was characterized by the presence of two additional absorption bands in the region around g=6, with respect to pH 6.5. Chloride addition resulted in the loss of these features and in a lower rhombicity of the signal. A unique EPR band at g=6.0 was obtained as a result of the interaction between HRP and dihydrogenphosphate, both in the absence and presence of 40 mM Cl −. We suggest that a synergistic effect of low pH, Cl − and ionic strength is responsible for dramatic modifications of the enzyme conformation consistent with the Fe(III)–His170 bond cleavage. Dihydrogenphosphate as well as high chloride concentrations are shown to display an unspecific effect, related to ionic strength. A mechanistic explanation for the acid transition of HRP, previously observed by Smulevich et al. [Biochemistry 36 (1997) 640] and interpreted as a pure pH effect, is proposed.