The role of oxoammonium cation in the SOD-Mimic activity of cyclic nitroxides

Cyclic nitroxides (RNO.) mimic the activity of superoxide dismutase (SOD) and demonstrate antioxidant properties in numerous in vitro and in vivo models. Their broad antioxidant activity may involve the participation of their reduced and oxidized forms, that is, hydroxylamine (RNO-H) and oxoammonium cation (RNO+). To examine this possibility we studied the reactions of RNO+ and RNO+ with HO2./O-2(.-) and with several reductants by pulse radiolysis and rapid-mixing stopped-flow techniques. The oxoammonium cations were generated by electrochemical and radiolytic oxidation of 2,2,6,6-tetramethylpiperidinoxyl (TPO) and 3-carbamoyl-2,2,5,5-tetramethylpyrrolidinoxyl (3-CP). The rate constant for the reaction of RNO. with HO2. to form RNO+ was determined to be (1.2 +/- 0.1) x 10(8) for TPO and (1.3 +/- 0.1) x 10(6) M-1 s(-1) for 3-CP. The kinetics results demonstrate that the reaction of RNO. with HO2. proceeds via an inner-sphere electron-transfer mechanism. The rate constant for the reaction of RNO. with O-2(.-) is lower than 1 x 10(3) M-1 s(-1). The rate constant for the reaction of RNO+ with O-2(.-) was determined to be (3.4 +/- 0.2) x 10(9) for TPO+ and (5.0 +/- 0.2) x 10(9) M-1 s(-1) for 3-CP+. Hence, both nitroxides catalyze the dismutation of superoxide through the RNO./RNO+ redox couple, and the dependence of the catalytic rate constant, k(cat), on pH displayed a bell-shaped curve having a maximum around pH 4. The oxoammonium cation oxidized ferrocyanide and HO2- by a one-electron transfer, whereas the oxidation of methanol, formate, and NADH proceeded through a two-electron-transfer reaction. The redox potential of RNO./RNO+ couple was calculated to be 0.75 and 0.89 V for 3-CP and TPO, respectively. The elucidated mechanism provides a clearer insight into the biological antioxidant properties of cyclic nitroxides that should permit design of even more effective antioxidants.