Active Site Conformations of Horseradish Peroxidase as Revealed by Time Resolved Resonance Raman Spectroscopy

By examining resonance Raman spectra of compound II of native, diacetyldeuteroheme-and manganese(Mn)-substituted horseradish peroxidase at different pHs, a metal-oxygen stretching vibration was found to be sensitive to the active site environment. At pH 7.0, compound II of diacetylheme isoenzyme C which is less reactive than native isoenzyme C showed an Fe=0 stretching vibration at 781 cm-1 which was 9 cm-1 higher than that of native one. At pH 10.0, the reactivities of compound II of native and diacetyl isoenzyme C become sluggish and Fe=0 stretching vibrations of compound II of both enzymes were found at about 790 cm-1. It is considered that a protonated amino acid residue which forms a hydrogen bonding to Fe=0 at pH 7.0 is responsible for the reactivity of compound II. In the ferrous-CO complex of native and deuteroheme horseradish peroxidase, we have found that the same protonated residue induced a tilt of the bound CO moiety with respect to the heme plane at pH 7.0.