Peroxynitrite modification of glutathione reductase: modeling studies and kinetic evidence suggest the modification of tyrosines at the glutathione disulfide binding site

The catalytic properties of glutathione reductase for its substrate,glutathione disulfide, were altered following a 60 s exposureto a 100-fold molar excess of peroxynitrite; the K_M value wasincreased by {small tilde}2.5-fold and the V_max value was decreasedby {small tilde}1.7-fold. The kinetic alterations are thoughtto result from nitrotyrosine formation as the intrinsic Tyrfluorescence is diminished. The UV-visible spectrum of glutathionereductase exhibited absorbance at {small tilde}423 nm, characteristicof nitrotyrosine. In addition, the presence of nitrotyrosinehas been detected by Western immunoblots with an anti-nitrotyrosineantibody. The peroxynitrite-induced inactivation is not observedin the presence of excess glutathione disulfide. However, excessNADPH offered no protection against peroxynitriteinduced inactivation.These observations suggest that the modification of {small tilde}1.8Tyr per subunit, at or near the glutathione disulfide bindingdomain, probably results in the observed catalytic alterations.To test this hypothesis, the two tyrosines closest to the glutathionedisulfide binding domain (Tyr114 and Tyr106), as indicated bythe X-ray crystallographic data Karplus and Schulz (1989) J.Biol. Chem, 210,163–180], were each converted to nitrotyrosinesby molecular modeling and the structure energy was minimized.These theoretical calculations indicate that the bond lengthsbetween Tyr114-O and the Gly-N and Cys II-N of glutathione disulfidebound to glutathione reductase (Karplus and Schulz, 1989) increasedby 3.0 and 4.3 Å, respectively, upon nitration. In thecase of Tyr106, the O–Cys II-N distance also increasesby {small tilde}1.6 Å. The loss of these hydrogen bondingcontacts is likely to result in the observed catalytic alterationsupon reaction with peroxy-nitrite.