Homolytic Pathways Drive Peroxynitrite-Dependent Trolox C Oxidation

摘要

Peroxynitrite is a powerful oxidant implicated as a mediator in nitric oxide(NO)-and superoxide(O_2~(centre dot-))-dependent toxicity.Peroxynitrite homolyzes after(i)protonation,yielding hydroxyl(OH)and nitrogen dioxide(centre dotNO_2)free radicals,and(ii)reaction with carbon dioxide(CO_2),yielding carbonate radical anion(CO_3~(centre dot-))and NO_2.Additionally,peroxynitrite reacts directly with several biomolecules.It is currently accepted that a-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 a-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 approx60 and approx31%,respectively,and increased to approx73 and approx40%,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~(centre dot-)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.