Glutathione Peroxidase Protects against Peroxynitrite-mediated Oxidations

Abstract

There is a requirement for cellular defense against excessive peroxynitrite generation to protect against DNA strand breaks and mutations and against interfer- ence with protein tyrosine-based signaling and other protein functions due to formation of 3-nitrotyrosine. Here, we demonstrate a role of selenium-containing en- zymes catalyzing peroxynitrite reduction using gluta- thione peroxidase (GPx) as an example. GPx protected against the oxidation of dihydrorhodamine 123 by per- oxynitrite more effectively than ebselen (2-phenyl-1,2- benzisoselenazol-3(2H)-one), a selenoorganic compound exhibiting a high second-order rate constant for the reaction with peroxynitrite, 2  106 M1 s1. Carboxy- methylation of selenocysteine in GPx by iodoacetate led to the loss of “classical” glutathione peroxidase activity but maintained protection against peroxynitrite-medi- ated oxidation. The maintenance of protection by GPx against peroxynitrite requires GSH as reductant. When peroxynitrite was infused to maintain a 0.2 M steady-state concentration, GPx in the presence of GSH, but neither GPx nor GSH alone, effectively inhibited the hydroxylation of benzoate by peroxynitrite. Under these steady-state conditions peroxynitrite did not cause the loss of classical GPx activity. GPx, like selenomethi- onine, protected against protein 3-nitrotyrosine forma- tion in human fibroblast lysates, shown in Western blots. The formation of nitrite rather than nitrate from peroxynitrite was enhanced by GPx or by selenomethi- onine. The results demonstrate a novel function of GPx GPx and potentially of other selenoproteins containing sel- enocysteine or selenomethionine, in the GSH-dependent maintenance of a defense line against peroxynitrite- mediated oxidations, as a peroxynitrite reductase.