Reaction of cyclic nitroxides with nitrogen dioxide: The intermediacy of the oxoammonium cations

Piperidine and pyrrolidine nitroxides, such as 2,2,6,6-tetramethylpiperidinoxyl (TPO) and 3-carbamoylproxyl (3-CP), respectively, are cell-permeable stable radicals, which effectively protect cells, tissues, isolated organs, and laboratory animals from radical-induced damage. The kinetics and mechanism of their reactions with OH, superoxide, and carbon-centered radicals have been extensively studied, but not with NO2, although the latter is a key intermediate in cellular nitrosative stress. In this research, NO2 was generated by pulse radiolysis, and its reactions with TPO, 4-OH-TPO, 4-oxo-TPO, and 3-CP were studied by fast kinetic spectroscopy, either directly or by using ferrocyanide or 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate), which effectively scavenge the product of this reaction, the oxoammonium cation. The rate constants for the reactions of NO2 With these nitroxides were determined to be (7-8) x 10(8) M-1 s(-1), independent of the pH over the range 3.9-10.2. These are among the highest rate constants measured for NO2 and are close to that of the reaction of NO2 with NO, that is, 1.1 x 10(9) M-1 s(-1). The hydroxylamines TPO-H and 4-OH-TPO-H are less reactive toward NO2, and an upper limit for the rate constant for these reactions was estimated to be 1 x 10(5) M-1 s(-1). The kinetics results demonstrate that the reaction of nitroxides with NO2 proceeds via an inner-sphere electron-transfer mechanism to form the respective oxoammonium cation, which is reduced back to the nitroxide through the oxidation of nitrite to NO2. Hence, the nitroxide slows down the decomposition of NO2 into nitrite and nitrate and could serve as a reservoir of NO2 unless the respective oxoammonium is rapidly scavenged by other reductant. This mechanism can contribute toward the protective effect of nitroxides against reactive nitrogen-derived species, although the oxoammonium cations themselves might oxidize essential cellular targets if they are not scavenged by common biological reductants, such as thiols.