Proton nuclear magnetic resonance studies of compounds I and II of horseradish peroxidase

Abstract

Proton high resolution nuclear magnetic resonance spectra have been successfully obtained for compounds I and II of horseradish peroxidase ( HRP ) at various temperatures and pH. The paramagnetically shifted peaks of heme peripheral methyl protons were well resolved to show a high and a low spin hemes having ferryl irons for compounds I and II, respectively. The spectrum was also observed in solution of horse metmyoglobin to which H 2O 2 was added. The electronic formulations of the hemes in compound II of HRP and “ferryl” myoglobin appear to be just alike as judged from their spectra. It is revealed from the proton spectra that the π-cation radical on the heme ring can be ruled out as a source of oxidizing equivalent in compound I of HRP. The pH dependent shifts for these intermediates afforded an evidence for a heme-linked ionizable group with pK=5.6 as the case for the native ferric HRP.