Nitration of γ-tocopherol and oxidation of α-tocopherol by copper-zinc superoxide dismutase/H(2)O(2)/NO(2)(−): Role of nitrogen dioxide free radical

Copper-zinc superoxide dismutase (Cu,ZnSOD) is the antioxidant enzyme that catalyzes the dismutation of superoxide (O(2)(•−)) to O(2) and H(2)O(2). In addition, Cu,ZnSOD also exhibits peroxidase activity in the presence of H(2)O(2), leading to self-inactivation and formation of a potent enzyme-bound oxidant. We report in this study that lipid peroxidation of l-α-lecithin liposomes was enhanced greatly during the SOD/H(2)O(2) reaction in the presence of nitrite anion (NO(2)(−)) with or without the metal ion chelator, diethylenetriaminepentacetic acid. The presence of NO(2)(−) also greatly enhanced α-tocopherol (α-TH) oxidation by SOD/H(2)O(2) in saturated 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine liposomes. The major product identified by HPLC and UV-studies was α-tocopheryl quinone. When 1,2-diauroyl-sn-glycero-3-phosphatidylcholine liposomes containing γ-tocopherol (γ-TH) were incubated with SOD/H(2)O(2)/NO(2)(−), the major product identified was 5-NO(2)-γ-TH. Nitrone spin traps significantly inhibited the formation of α-tocopheryl quinone and 5-NO(2)-γ-TH. NO(2)(−) inhibited H(2)O(2)-dependent inactivation of SOD. A proposed mechanism of this protection involves the oxidation of NO(2)(−) by an SOD-bound oxidant to the nitrogen dioxide radical ((•)NO(2)). In this study, we have shown a new mechanism of nitration catalyzed by the peroxidase activity of SOD. We conclude that NO(2)(−) is a suitable probe for investigating the peroxidase activity of familial Amyotrophic Lateral Sclerosis-linked SOD mutants.