Structural implication of the heme-linked ionization of horseradish peroxidase probed by the Fe-histidine stretching Raman line.

Resonance Raman spectra of horseradish peroxidase (HRP) in the 200 to 600 cm" region were measured for the reduced and oxidized states and in the presence and absence of substrate, For the reconstituted HRP with a S4Fe-substituted heme, the Raman line of ferro HRP at 244 cm" was shifted to 246 cm" but all other modes remained unshifted. The amount of the frequency shift agreed with that observed for the Fe-N,(His F8) stretching vibration of deoxy Mb. Therefore, the 244-cm" line of ferro HRP was assigned to the mode primarily involving the Fe-N,(His, proximal) stretching mode. Only this mode exhibited a pH-dependent frequency shift by 3 to 5 cm" toward lower frequency at higher pH with the midpoint pH value at 7 for isoenzyme C and 5.5 for isoenzyme A2. The p& values were in good agreement with those observed by other methods for these HRP isoenzymes and were ascribed to the heme-linked ionization of an amino acid residue. The corresponding mode of deoxy Mb and Fe(II)(protoporphyrin IX) 2methylimidazole (2-MeIm) did not show a sign of the pH-dependent frequency shift. On the other hand, the Fe-N(2-MeIm) stretching frequency of Fe(II)(protoporphyrin IX)(2-MeIm) was lowered upon incorporation of it into a micelle of detergent and further lowered in an organic solvent. The stretching frequency was raised when the coordinated 2-MeIm was ionized, and the frequency change amounted nearly to the difference between the Fe-N,(His, proximal) stretching frequencies of deoxy Mb and ferro HRP, whereas it was much larger than the pH-dependent frequency shift of ferro HRP. The upward frequency shift upon ionization of 2-MeIm contrasted with the downward shift of the Fe-N,(His) stretching frequency of ferro HRP at alkaline side and hence was not compatible with the idea that the proximal histidine was deprotonated upon the heme-linked ionization. It seems rather likely that the proximal histidine is very strongly hydrogen-bonded at both sides of the transition but an appreciable strain is exerted to the Fe-N,(His) bond at the alkaline side presumably due to a conformation change of the polypeptide chain upon ionization of distal histidine. The Fe-N,(His) stretching mode of ferri HRP appeared at 274 cm". This mode also showed a pH-dependent frequency shift in accord with the transition reported at pH 11. The Raman spectrum of ferri HRP in the higher frequency region indicated a change of coordination number of the heme iron upon binding of benzhydroxamic acid but no change upon binding of hydroquinone.