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.
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 (lambda=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 (lambda=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(II)-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.
Laurenti
E
E
Dipartimento di Chimica I.F.M., Università di Torino, Italy.
Suriano
G
G
Ghibaudi
E M
EM
Ferrari
R P
RP
D002712:Chlorides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry*; D007501:Iron / Q000737:chemistry*; D009994:Osmolar Concentration; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D002712:Chlorides; D007093:Imidazoles; D006418:Heme; C029899:imidazole; D007501:Iron; D006735:Horseradish Peroxidase