Homolytic pathways drive peroxynitrite-dependent Trolox C oxidation.

Peroxynitrite is a powerful oxidant implicated as a mediator in nitric oxide ((*)NO)- and superoxide (O(2)(*)(-))-dependent toxicity. Peroxynitrite homolyzes after (i) protonation, yielding hydroxyl ((*)OH) and nitrogen dioxide ((*)NO(2)) free radicals, and (ii) reaction with carbon dioxide (CO(2)), yielding carbonate radical anion (CO(3)(*)(-)) and (*)NO(2). Additionally, peroxynitrite reacts directly with several biomolecules. It is currently accepted that alpha-tocopherol is one important antioxidant in lipid compartments and its reactions with peroxynitrite or peroxynitrite-derived radicals may be relevant in vivo. Previous reports on the peroxynitrite reaction with Trolox C (TxOH)--an alpha-tocopherol water soluble analogue--suggested a direct and fast reaction. This was unexpected to us as judged from the known reactivities of peroxynitrite with other phenolic compounds; thus, we thoroughly investigated the kinetics and mechanism of the reaction of peroxynitrite with TxOH and its modulation by CO(2). Direct electron paramagnetic resonance studies revealed that Trolox C phenoxyl radical (TxO(*)) was the only paramagnetic species detected either in the absence or in the presence of CO(2). Stopped-flow spectrophotometry experiments revealed a sequential reaction mechanism, with the intermediacy of TxO(*) and the production of Trolox C quinone (TxQ). Reactions were zero-order with respect to TxOH and first-order in peroxynitrite and CO(2), demonstrating that the reaction of peroxynitrite with TxOH is indirect. In agreement, TxOH was unable to inhibit the direct peroxynitrite-mediated oxidation of methionine to methionine sulfoxide. TxOH oxidation yields to TxO(*) and TxQ with respect to peroxynitrite were approximately 60 and approximately 31%, respectively, and increased to approximately 73 and approximately 40%, respectively, in the presence of CO(2). At peroxynitrite excess over TxOH, the kinetics and mechanism of oxidation are more complex and involve the reactions of CO(3)(*)(-) with TxO(*) and the possible intermediacy of unstable NO(2)-TxOH adducts. Taken together, our results strongly support that H(+)- or CO(2)-catalyzed homolysis of peroxynitrite is required to cause TxOH, and hence, alpha-tocopherol oxidation.