Dopamine Prevents Nitration of Tyrosine Hydroxylase by Peroxynitrite and Nitrogen Dioxide

Abstract

Peroxynitrite and nitrogen dioxide (NO 2 ) are reactive nitrogen species that have been implicated as causal factors in neurodegenerative conditions. Peroxynitriteinduced nitration of tyrosine residues in tyrosine hydroxylase (TH) may even be one of the earliest biochemical events associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced damage to dopamine neurons. Exposure of TH to peroxynitrite or NO 2 results in nitration of tyrosine residues and modification of cysteines in the enzyme as well as inactivation of catalytic activity. Dopamine (DA), its precursor 3,4-dihydroxyphenylalanine, and metabolite 3,4-dihydroxyphenylacetic acid completely block the nitrating effects of peroxynitrite and NO 2 on TH but do not relieve the enzyme from inhibition. o-Quinones formed in the reaction of catechols with either peroxynitrite or NO 2 react with cysteine residues in TH and inhibit catalytic function. Using direct, real-time evaluation of tyrosine nitration with a green fluorescent protein-TH fusion protein stably expressed in intact cells (also stably expressing the human DA transporter), DA was also found to prevent NO 2 -induced nitration while leaving TH activity inhibited. These results show that peroxynitrite and NO 2 react with DA to form quinones at the expense of tyrosine nitration. Endogenous DA may therefore play an important role in determining how DA neurons are affected by reactive nitrogen species by shifting the balance of their effects away from tyrosine nitration and toward o-quinone formation.Peroxynitrite (ONOO Ϫ ) 1 is formed in the chemical reaction between nitric oxide and superoxide (1). ONOO Ϫ is a powerful oxidant that can modify proteins and cell organelles, damage DNA, and cause lipid peroxidation, properties that are thought to underlie its cytotoxicity (2, 3). ONOO Ϫ nitrates free tyrosine and tyrosine residues in proteins, modifications that are used as markers of ONOO Ϫ action under conditions of cellular damage and in numerous diseases (4, 5). ONOO Ϫ has been implicated as a causal factor in dopamine (DA) neuronal damage that occurs after exposure to the neurotoxic amphetamines (6 -8) and MPTP (9, 10). It is also suspected to play a role in the etiology of idiopathic Parkinson's disease (11,12).The participation of ONOO Ϫ in cellular or neuronal toxicity is an evolving and complicated issue. Real-time measures of intracellular nitration indicate that ONOO Ϫ may not cross cell membranes in sufficient amounts to cause intracellular tyrosine nitration (13). This may be a reflection of preferential nitration of hydrophobic, transmembrane tyrosines by ONOO Ϫ as compared with tyrosines in the aqueous phase (14). Mayer and colleagues (15) have argued on chemical and kinetic grounds that ONOO Ϫ is altogether ineffective as a tyrosine nitrating species in vivo. ONOO Ϫ is by no means the only nitrating species and a strong case can be made for nitrogen dioxide (NO 2 ) as a more relevant nitrating reagent (13, 16).The tyrosine-nitrating properties of ONOO Ϫ and NO 2 are not often cons...

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