Heme-modification studies on horseradish peroxidase

Abstract

Synthetic horseradish peroxidases (donor: H 2O 2 oxidoreductase, EC 1.11.1.7) were prepared from apoenzyme and modified heme. The synthetic enzymes containing proto-, meso- or deuteroheme or protoheme monomethyl ester reacted with stoichiometric amounts of hydroperoxides, to form Compound I with these synthetic enzymes. Other hydroperoxide-induced compounds such as Compounds II and III could also be formed with these synthetic enzymes. The optical spectra of these compounds were similar to those of the native enzyme, except for shifts in absorption maxima. The enzyme which contained protoheme dimethyl ester did not form these compounds under the same experimental conditions. Except for the protoheme dimethyl ester-containing enzyme, these synthetic enzymes reacted with heme ligands such as fluoride, cyanide, and carbon monoxide to form well-defined complexes and were converted into alkaline forms on raising the pH. The affinities of the enzyme for these ligands were similar to those of native enzyme. The pH at which the alkaline transition occurred was similar to that for the native enzyme, except for the protoheme monomethyl ester-containing enzyme which showed a considerably lower p K value. The enzymatic activities of the synthetic enzymes which contained meso-, deuterohemes in the oxidation of guaiacol were similar to that of the native enzyme. The enzyme which contained protoheme monomethyl ester showed only 20% of the activity of native enzyme, and the enzyme containing protoheme dimethyl ester showed no peroxidase activity. It was concluded that the side chains at positions 2 and 4 of the porphyrin ring were not essential for the catalytic activity of horseradish peroxidase, but that the free carboxyl groups at positions 6 and 7 were essential.