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 exposure to a 100-fold molar excess of peroxynitrite; the K(M) value was increased by approximately 2.5-fold and the V(max) value was decreased by approximately 1.7-fold. The kinetic alterations are thought to result from nitrotyrosine formation as the intrinsic Tyr fluorescence is diminished. The UV-visible spectrum of glutathione reductase exhibited absorbance at approximately 423 nm, characteristic of nitrotyrosine. In addition, the presence of nitrotyrosine has been detected by Western immunoblots with an anti-nitrotyrosine antibody. The peroxynitrite-induced inactivation is not observed in the presence of excess glutathione disulfide. However, excess NADPH offered no protection against peroxynitrite-induced inactivation. These observations suggest that the modification of approximately 1.8 Tyr per subunit, at or near the glutathione disulfide binding domain, probably results in the observed catalytic alterations. To test this hypothesis, the two tyrosines closest to the glutathione disulfide binding domain (Tyr114 and Tyr106), as indicated by the X-ray crystallographic data [Karplus and Schulz (1989) J. Biol. Chem., 210, 163-180], were each converted to nitrotyrosines by molecular modeling and the structure energy was minimized. These theoretical calculations indicate that the bond lengths between Tyr114-O and the Gly-N and Cys II-N of glutathione disulfide bound to glutathione reductase (Karplus and Schulz, 1989) increased by 3.0 and 4.3 A, respectively, upon nitration. In the case of Tyr106 the 0-Cys II-N distance also increases by approximately 1.6 A. The loss of these hydrogen bonding contacts is likely to result in the observed catalytic alterations upon reaction with peroxynitrite.