XXXV Reunião Anual da SBBqResumoID:9575


Protein radical formation and tyrosine nitration in human SOD in presence of simultaneous fluxes of nitric oxide and superoxide.
Demicheli, V.1; Quijano, C.1; Alvarez, B.2 and Radi, R.1

1 Depto. Bioquímica, Facultad de Medicina and 2 Laboratorio Enzimología, Facultad de Ciencias, Universidad de la República, Uruguay.

Human recombinant MnSOD and CuZnSOD were exposed to simultaneous fluxes of superoxide (JO2.-) and nitric oxide (J.NO) (10 mM/min, 10 min) and the product of these radicals, peroxynitrite, was determined to be responsible for the formation of protein-derived radicals as detected by immuno-spin trapping, using an anti-DMPO nitrone antibody. The formation of protein radicals was followed by tyrosine (Tyr) nitration in MnSOD, and, in both Cu,Zn- and MnSOD the exposition to the fluxes led to a time-dependent inactivation. The SODs were also exposed to J.NO and JO2.- in presence of scavengers of peroxynitrite and peroxynitrite-derived free radicals, in order to determine the nature of the nitrating and inactivating species. Uric acid decreased nitration but it did not prevent from inactivation. On the other hand, glutathione (GSH) partially inhibited both inactivation and Tyr nitration. The inhibitory effects of GSH, however, were total if authentic peroxynitrite was added either as a bolus or flux. The data is consistent with the previously reported metal-catalyzed nitration of Tyr-34 in MnSOD. Since both, uric acid and GSH, readily react with nitrogen dioxide we propose that in the presence of these compounds only the site-specific nitration at the active site of the enzyme is observed. Moreover, and alternatively, tyrosyl-34 radical might evolve to iminoxyl radical by reaction with nitric oxide followed by its oxidation to 3-nitrotyrosine. Our results could explain MnSOD tyrosine nitration observed in vivo in inflammatory conditions in presence of low molecular weight scavengers such as GSH.