Homolytic Pathways Drive Peroxynitrite-Dependent Trolox C Oxidation

Peroxynitrite is a powerful oxidant implicated as a mediator in nitric oxide (^•NO)- and superoxide (O₂^•-)-dependent toxicity. Peroxynitrite homolyzes after (i) protonation, yielding hydroxyl (^•OH) and nitrogen dioxide (^•NO₂) free radicals, and (ii) reaction with carbon dioxide (CO₂), yielding carbonate radical anion (CO₃^•-) and ^•NO₂. Additionally, peroxynitrite reacts directly with several biomolecules. It is currently accepted that α-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 α-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 ∼60 and ∼31%, respectively, and increased to ∼73 and ∼40%, 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-catalyzed homolysis of peroxynitrite is required to cause TxOH, and hence, α-tocopherol oxidation.