Hydroxylation of p-coumaric acid by horseradish peroxidase. The role of superoxide and hydroxyl radicals.

1. In the presence of dihydroxyfumarate, horseradish peroxidase catalyses the conversion of p-coumaric acid into caffeic acid at pH 6. This hydroxylation is completely inhibited by superoxide dismutase. 2. Dihydroxyfumarate cannot be replaced by ascorbate H2O2, NADH, cysteine or sulphite. Peroxidase can be replaced by high (10 mM) concentrations of FeSO4, but this reaction is almost unaffected by superoxide dismutase. 3. Hydroxylation by the peroxidase/dihydroxyfumarate system is completely inhibited by low concentrations of Mn2+ or Cu2+. It is proposed that this is due to the ability of these metal ions to react with the superoxide radical O2--. 4. Hydroxylation is partially inhibited by mannitol, Tris or ethanol and completely inhibited by formate. This seems to be due to the ability of these reagents to react with the hydroxyl radical -OH. 5. It is concluded that O2-- is generated during the oxidation of dihydroxyfumarate by peroxidase and reacts with H2O2 to produce hydroxyl radicals, which then convert p-coumaric acid into caffeic acid.