Homolytic pathways drive peroxynitrite-dependent trolox C oxidation

Peroxynitrite is a powerful oxidant implicated as a mediator in nitric oxide ((NO)-N-.)- and superoxide (O-2(.-))-dependent toxicity. Peroxynitrite homolyzes after (i) protonation, yielding hydroxyl ((OH)-O-.) and nitrogen dioxide ((NO2)-N-.) free radicals, and (ii) reaction with carbon dioxide (CO2), yielding carbonate radical anion (CO3.-) and (NO2)-N-.. Additionally, peroxynitrite reacts directly with several biomolecules. It is currently accepted that (x-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 CO2. 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 CO2. 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 CO2, 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 similar to60 and similar to31%, respectively, and increased to similar to73 and similar to40%, respectively, in the presence of CO2. At peroxynitrite excess over TxOH, the kinetics and mechanism of oxidation are more complex and involve the reactions Of CO3.- with TxO(.) and the possible intermediacy of unstable NO2-TxOH adducts. Taken together, our results strongly support that H+- or CO2- atalyzed homolysis of peroxynitrite is required to cause TxOH, and hence, alpha-tocopherol oxidation.