Raman spectroscopic studies of compound II of horseradish peroxidase show that the oxygen atom in the FeIV = O group of the heme is rapidly exchanged in H2O at pH 7.0 but not in an alkaline solution (pH 11.0). This conclusion is based on studies of shift in the FeIV = O stretching mode of compound II in H2(18)O; further studies show that the FeIV = O heme is hydrogen-bonded to an amino acid residue of the protein in neutral solutions but not in the alkaline solution. Deprotonation of this residue takes place with the midpoint pH at 8.8 and accordingly corresponds to the so-called heme-linked ionization. It is concluded that this hydrogen-bonded proton plays an important part in the oxygen exchange mechanism. From this it seems clear that this hydrogen-bonded proton has an essential role in the acid/base catalysis of this enzyme and that alkaline deactivation of this enzyme can be attributed to the lack of a hydrogen-bonded proton at high pH.