SUPEROXIDE AND PEROXIDASE-CATALYSED REACTIONS. OXIDATION OF DIHYDROXYFUMARATE, NADH AND DITHIOTHREITOL BY HORSERADISH PEROXIDASE*

Abstract— When oxidizing NADH or dihydroxyfumarate, horseradish peroxidase exists mainly as compound III, which is formed by a reaction between O ‐ 2 and the enzyme. Certain phenols stimulate peroxidase‐catalysed oxidation of NADH or dihydroxyfumarate, apparently because they bring about the breakdown of compound III to active enzyme. Superoxide dismutase and catalase, added at any time during the reaction, inhibit dihydroxyfumarate oxidation by peroxidase. This is attributed to generation of the dihydroxyfumaryl radical during H 2 O 2 ‐dependent oxidation of dihydroxyfumarate by peroxidase. This radical reduces O 2 to O ‐ 2 , which can then react with more dihydroxyfumarate. Superoxide dismutase added during the course of the reaction does not inhibit NADH oxidation by peroxidase, since O ‐ 2 , although generated in the reaction mixture, reacts only slowly with NADH. Lactate dehydrogenase or Mn 2+ stimulate NADH oxidation by peroxidase because they catalyse a reaction between NADH and O ‐ 2 . Oxidation of NADH by peroxidase in the presence of certain phenols and Mn 2+ is very rapid, and requires both H 2 O 2 and O ‐ 2 . O ‐ 2 also seems to be necessary to initiate NADH oxidation by peroxidase. Aromatic compounds are hydroxylated by a mixture of peroxidase and dihydroxyfumarate. Hydroxylation is prevented by superoxide dismutase or by scavengers of the hydroxy radical (.OH). O ‐ 2 is insufficiently reactive to hydroxylate aromatic rings directly and so may give rise to .OH, which is the true hydroxylating species. Hydroxyl radical probably does not arise by the Haber‐Weiss reaction. H 2 O 2 , but not O ‐ 2 , is involved in the oxidation of thiol compounds by peroxidase.