Hydroxyl-radical production in physiological reactions - A novel function of peroxidase

Peroxidases catalyze the dehydrogenation by hydrogen peroxide (H2O2) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of O-2 to superoxide (O-2(.-)) and H2O2 by Substrates such as NADH or dihydroxyfumarate. Here we show that horseradish peroxidase can also catalyze a third type of reaction that results in the production of hydroxyl radicals ((OH)-O-.) from H2O2 in the presence of O-2(.-) We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by O-2(.-) into the perferryl form (Compound III), in which the haem iron can assume the ferrous state. It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of (OH)-O-. from O-2(.-) and H2O2 (iron-catalyzed Haber-Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. O-2(.-) and H2O2 can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The (OH)-O-.-producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various O-2(.-) and (OH)-O-. scavengers. On an equimolar Fe basis, horseradish peroxidase is 1-2 orders of magnitude more active than Fe-EDTA, an inorganic catalyst of the Haber-Weiss reaction. Particularly high (OH)-O-.-producing activity was found in the alkaline horseradish peroxidase isoforms and in a ligninase-type fungal peroxidase, whereas lactoperoxidase and soybean peroxidase were less active, and myeloperoxidase was inactive. Operating in the (OH)-O-.-producing mode, peroxidases may be responsible for numerous destructive and toxic effects of activated oxygen reported previously.