Protein control of prosthetic heme reactivity. Reaction of substrates with the heme edge of horseradish peroxidase

Abstract

Incubation of horseradish peroxidase with phenylhydrazine and H2O2 markedly depresses the catalytic activity and the intensity, but not position, of the Soret band. Approximately 11-13 mol of phenylhydrazine and 25 mol of H2O2 are required per mol of enzyme to minimize the chromophore intensity. The enzyme retains some activity after such treatment, but this activity is eliminated if the enzyme is isolated and reincubated with phenylhydrazine. The prosthetic heme of the enzyme does not react with phenylhydrazine to give a sigma-bonded phenyl-iron complex, as it does in other hemoproteins, but is converted instead to the delta-mesophenyl and 8-hydroxymethyl derivatives. The loss of activity is due more to protein than heme modification, however. The inactivated enzyme reacts with H2O2 to give a spectroscopically detectable Compound I. The results imply that substrates interact with the heme edge rather than with the activated oxygen of Compounds I and II and specifically identify the region around the delta-meso-carbon and 8-methyl group as the exposed sector of the heme. Horseradish peroxidase, in contrast to cytochrome P-450, generally does not catalyze oxygen-transfer reactions. The present results indicate that oxygen-transfer reactions do not occur because the activated oxygen and the substrate are physically separated by a protein-imposed barrier in horseradish peroxidase.