| Tyrosine nitration has been observed in human diseases associated with oxidative stress. The pathways leading to nitration of tyrosine residues in vivo are still unclear. Studies have shown that peroxynitrite anion can be responsible for nitration of proteins. Alternatively, tyrosine nitration may be mediated nitrogen dioxide produced by oxidation of nitrite, catalyzed by peroxidases. Here, we studied the possible mechanisms and selectivity of nitration of two proteins known to be nitrated in vivo, manganese superoxide dismutase (MnSOD) and alpha-synuclein. MnSOD, which plays a critical role in cellular defense against oxidative stress, is found to be nitrated and inactive under disease conditions. We determined the mechanism of the reaction between MnSOD and peroxynitrite that results in nitration; the patterns of MnSOD nitration by various reagents were also investigated. We established that the residues Tyr34, Tyr9, and Tyr11 were most susceptible to nitration, and the relative amount of nitration of these residues depended upon the nature of the nitration agent. Nitration mediated by peroxynitrite and carbonate radical anion resulted in nitration of Tyr34, while nitration by nitrogen dioxide led to Tyr9 and Tyr11 nitration. We also observed that only nitration of Tyr34 caused inactivation of the enzyme; we conclude that the loss of activity of nitrated MnSOD in vivo shows that the responsible reagent is peroxynitrite. We also studied the selectivity of alpha-synuclein nitration, which has been observed in the signature inclusions of Parkinson's disease. We determined that there is a subtle selectivity towards nitration of the residue Tyr39, which did not depend on the agent of nitration or the presence of lipids. Since, the residue Tyr39 is thought to involved in the possible lipid binding role of a-synuclein in vivo, this selectivity might have profound influence on the development of Parkinson's disease. |
