Abstract
There is a requirement for cellular defense against excessive peroxynitrite generation to protect against DNA strand breaks and mutations and against interference with protein tyrosine-based signaling and other protein functions due to formation of 3-nitrotyrosine. Here, we demonstrate a role of selenium-containing enzymes catalyzing peroxynitrite reduction using glutathione peroxidase (GPx) as an example. GPx protected against the oxidation of dihydrorhodamine 123 by peroxynitrite 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 x 10(6) M-1 s-1. Carboxymethylation of selenocysteine in GPx by iodoacetate led to the loss of "classical" glutathione peroxidase activity but maintained protection against peroxynitrite-mediated oxidation. The maintenance of protection by GPx against peroxynitrite requires GSH as reductant. When peroxynitrite was infused to maintain a 0.2 microM 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 selenomethionine, protected against protein 3-nitrotyrosine formation in human fibroblast lysates, shown in Western blots. The formation of nitrite rather than nitrate from peroxynitrite was enhanced by GPx or by selenomethionine. The results demonstrate a novel function of GPx and potentially of other selenoproteins containing selenocysteine or selenomethionine, in the GSH-dependent maintenance of a defense line against peroxynitrite-mediated oxidations, as a peroxynitrite reductase.
Highlights
There is a requirement for cellular defense against excessive peroxynitrite generation to protect against DNA strand breaks and mutations and against interference with protein tyrosine-based signaling and other protein functions due to formation of 3-nitrotyrosine
The results demonstrate a novel function of glutathione peroxidase (GPx) and potentially of other selenoproteins containing selenocysteine or selenomethionine, in the GSH-dependent maintenance of a defense line against peroxynitritemediated oxidations, as a peroxynitrite reductase
Protein tyrosine nitration may interfere with phosphorylation/ dephosphorylation signaling [3], and the in vivo occurrence of protein nitration in the human has been demonstrated in patients chronically rejecting renal allografts [4]. 3-Nitrotyrosine plasma levels of up to 0.12 mM were observed in chronic renal failure patients with septic shock [5]
Summary
There is a requirement for cellular defense against excessive peroxynitrite generation to protect against DNA strand breaks and mutations and against interference with protein tyrosine-based signaling and other protein functions due to formation of 3-nitrotyrosine. The results demonstrate a novel function of GPx and potentially of other selenoproteins containing selenocysteine or selenomethionine, in the GSH-dependent maintenance of a defense line against peroxynitritemediated oxidations, as a peroxynitrite reductase. Selenocysteine, and selenomethionine protected DNA from single-strand break formation caused by peroxynitrite more effectively than their sulfur-containing analogs [8]. These selenocompounds were protective in model oxidation and nitration reactions mediated by peroxynitrite [9]
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