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

Abstract

Copper-zinc superoxide dismutase (Cu,Zn-SOD) 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-␣-lecithin liposomes was enhanced greatly during the SOD͞H 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 ␣-tocopherol (␣-TH) oxidation by SOD͞H 2 O 2 in saturated 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine liposomes. The major product identified by HPLC and UVstudies was ␣-tocopheryl quinone. When 1,2-diauroyl-snglycero-3-phosphatidylcholine liposomes containing ␥-tocopherol (␥-TH) were incubated with SOD͞H 2 O 2 ͞NO 2 ؊ , the major product identified was 5-NO 2 -␥-TH. Nitrone spin traps significantly inhibited the formation of ␣-tocopheryl quinone and 5-NO 2 -␥-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.

Bicarbonate Enhances the Peroxidase Activity of Cu,Zn-Superoxide Dismutase

“…These small molecular weight anionic ligands are presumed to be oxidized by the oxidant formed at the active site of SOD1. The oxidizing potential of this putative SOD-Cu 2ϩ -⅐ OH species is similar to that of the "free" hydroxyl radical, which is considerably higher than those associated with conventional peroxidases (4). The one-electron oxidation potential for the HCO 3 Ϫ /carbonate radical anion (CO 3 . )…”

“…The molar extinction coefficient of the Cu 2ϩ -and Zn 2ϩ -PAR complex remained unchanged up to 250 mM HCO 3 Ϫ . ESR Measurements-The formation of ␣-tocopheroxyl (␣-T ⅐ ) and 5-nitro-␥-tocopheroxyl (NGT ⅐ ) radicals was monitored by ESR (4,20). ESR measurements were performed at ambient temperature using a Varian E-109 spectrometer operating at 9.5 GHz (X-band) and 100 kHz field modulation.…”

“…, although less reactive than the hydroxyl radical, is a more selective oxidant that may diffuse over a longer distance, thereby causing oxidative damage to distant biological targets (9,10). We measured the peroxidase activity of SOD1 under anaerobic conditions by using the nitrite anion (NO 2 Ϫ ) as a peroxidase substrate (4). The oxidation of NO 2 Ϫ to the nitrogen dioxide free radical (NO 2 ⅐ ) was measured by monitoring the formation of ␣-tocopheryl quinone (␣-TQ) and 5-nitro-␥-tocopherol (NGTH) (4,(11)(12)(13)(14).…”

“…We measured the peroxidase activity of SOD1 under anaerobic conditions by using the nitrite anion (NO 2 Ϫ ) as a peroxidase substrate (4). The oxidation of NO 2 Ϫ to the nitrogen dioxide free radical (NO 2 ⅐ ) was measured by monitoring the formation of ␣-tocopheryl quinone (␣-TQ) and 5-nitro-␥-tocopherol (NGTH) (4,(11)(12)(13)(14). Our results clearly demonstrate that the bicarbonate anion is not required for the peroxidase activity of SOD1; however, bicarbonate enhances oxidation of ␣-and ␥-tocopherols in the presence and absence of the nitrite anion.…”

“…Measurement of SOD1 Activity-SOD1 activity was measured using the ferricytochrome c (cyt c) reduction assay (4,17). Briefly, xanthine (0.5 mM) and xanthine oxidase (0.05 units/ml) were incubated with cyt c (20 M) in phosphate buffer (200 mM, pH 7.4).…”