Function of thioredoxin reductase as a peroxynitrite reductase using selenocystine or ebselen.

Abstract

The activity of mammalian thioredoxin reductase as a peroxynitrite reductase was investigated. Peroxynitrite was infused to maintain a 0.2 microM steady-state concentration in potassium phosphate buffer (pH 7.4). Benzoate hydroxylation and nitrite formation were used as indices of oxidation reactions of peroxynitrite and of peroxynitrite reduction, respectively. In the presence of NADPH (10 microM), thioredoxin reductase at 50 nM alone did not significantly scavenge peroxynitrite, as shown by there being no significant effect on benzoate hydroxylation or nitrite formation. However, when selenocystine (1 microM) or ebselen (2 microM) was present in the reaction mixture, there was significant suppression of benzoate hydroxylation and an increase in nitrite formation until all the NADPH was oxidized. The addition of thioredoxin did not enhance these effects. In contrast, peroxynitrite reduction by ebselen complexed with BSA was enhanced by the presence of thioredoxin. In parallel experiments, thioredoxin reductase efficiently reduced ebselen selenoxide back to ebselen.