Nitration and inactivation of tyrosine hydroxylase by peroxynitrite

Tyrosine hydroxylase (TH) is modified by nitration after exposure of mice t o 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine. The temporal associat ion of tyrosine nitration with inactivation of TH activity in vitro suggest s that this covalent post-translational modification is responsible for the in vivo loss of TH function (Ara, J., Przedborski, S., Naini, A. B., Jacks on-Lewis, V., Trifiletti, R. R., Horwitz, J., and Ischiropoulos, H. (1998) Proc. Natl. Acad Sci. U. S. A. 95, 7659 -7663). Recent data showed that cys teine oxidation rather than tyrosine nitration is responsible for TH inacti vation after peroxynitrite exposure in vitro (Kuhn, D. M., Aretha, C. W., a nd Geddes, T. J. (1999) J. Neurosci. 19, 10289-10294). However, re-examinat ion of the reaction of peroxynitrite with purified TH failed to produce cys teine oxidation but resulted in a concentration-dependent increase in tyros ine nitration and inactivation. Cysteine oxidation is only observed after p artial unfolding of the protein. Tyrosine residue 423 and to lesser extent tyrosine residues 428 and 432 are modified by nitration. Mutation of Tyr(42 3) to Phe resulted in decreased nitration as compared with wild type protei n without loss of activity. Stopped-flow experiments reveal a second order rate constant of (3.8 +/- 0.9) X 10(3) M-1 s(-1) at pH 7.4 and 25 degreesC for the reaction of peroxynitrite with TH. Collectively, the data indicate that peroxynitrite reacts with the metal center of the protein and results primarily in the nitration of tyrosine residue 423, which is responsible fo r the inactivation of TH.