A mechanistic study of the formation of hydroxyl radicals induced by horseradish peroxidase with NADH

During the oxidation of NADH by horseradish peroxidase (HRP-Fe3+), superoxide (O-2(-)) is produced, and HRP-Fe3+ is converted to compound III. Superoxide dismutase inhibited both the generation of O-2(-) and the formation of compound III. In contrast, catalase inhibited only the generation of O-2(-). Under anaerobic conditions, the formation of compound III did not occur in the presence of NADH, thus indicating that compound III is produced via formation of a ternary complex consisting of HRP-Fe3+, NADH and oxygen. The generation of hydroxyl radicals was dependent upon O-2(-) and H2O2 produced by HRP-Fe3+-NADH. The reaction of compound III with H2O2 caused the formation of compound II without generation of hydroxyl radicals. Only HRP-Fe3+-NADH (but not K+O2- and xanthine oxidase-hypoxanthine) was able to induce the conversion of metmyoglobin to oxymyoglobin, thus suggesting the participation of a ternary complex made up of HRP-Fe(2+center dot center dot center dot)O(2)(center dot center dot center dot)NAD(center dot) (but not free O-2(-) or H2O2) in the conversion of metmyoglobin to oxymyoglobin. It appears that a cyclic pathway is formed between HRP-Fe3+, compound III and compound II in the presence of NADH under aerobic conditions, and a ternary complex plays the central roles in the generation of O-2(-) and hydroxyl radicals.