Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
J Am Chem Soc. 2010 Dec 8;132(48):17174-85. doi: 10.1021/ja105684w. Epub 2010 Nov 16.
Protein tyrosine nitration has been observed in a variety of human diseases associated with oxidative stress, such as inflammatory, neurodegenerative, and cardiovascular conditions. However, the pathways leading to nitration of tyrosine residues are still unclear. Recent studies have shown that peroxynitrite (PN), produced by the reaction of superoxide and nitric oxide, can lead to protein nitration and inactivation. Tyrosine nitration may also be mediated by nitrogen dioxide produced by the oxidation of nitrite by peroxidases. Manganese superoxide dismutase (MnSOD), which plays a critical role in cellular defense against oxidative stress by decomposing superoxide within mitochondria, is nitrated and inactivated under pathological conditions. In this study, MnSOD is shown to catalyze PN-mediated self-nitration. Direct, spectroscopic observation of the kinetics of PN decay and nitrotyrosine formation (k(cat) = 9.3 × 10(2) M(-1) s(-1)) indicates that the mechanism involves redox cycling between Mn(2+) and Mn(3+), similar to that observed with superoxide. Distinctive patterns of tyrosine nitration within MnSOD by various reagents were revealed and quantified by MS/MS analysis of MnSOD trypsin digest peptides. These analyses showed that three of the seven tyrosine residues of MnSOD (Tyr34, Tyr9, and Tyr11) were the most susceptible to nitration and that the relative amounts of nitration of these residues varied widely depending upon the nature of the nitrating agent. Notably, nitration mediated by PN, in both the presence and absence of CO2, resulted in nitration of the active site tyrosine, Tyr34, while nitration by freely diffusing nitrogen dioxide led to surface nitration at Tyr9 and Tyr11. Flux analysis of the nitration of Tyr34 by PN-CO2 showed that the nitration rate coincided with the kinetics of the reaction of PN with CO2. These kinetics and the 20-fold increase in the efficiency of tyrosine nitration in the presence of CO2 suggest a specific role for the carbonate radical anion (•CO3(-)) in MnSOD nitration by PN. We also observed that the nitration of Tyr34 caused inactivation of the enzyme, while nitration of Tyr9 and Tyr11 did not interfere with the superoxide dismutase activity. The loss of MnSOD activity upon Tyr34 nitration implies that the responsible reagent in vivo is peroxynitrite, acting either directly or through the action of •CO3(-).
蛋白质酪氨酸硝化已在多种与氧化应激相关的人类疾病中观察到，如炎症、神经退行性和心血管疾病。然而，导致酪氨酸残基硝化的途径仍不清楚。最近的研究表明，过氧亚硝酸盐（PN），由超氧化物和一氧化氮反应产生，可导致蛋白质硝化和失活。酪氨酸硝化也可能由过氧化物酶氧化亚硝酸盐产生的二氧化氮介导。锰超氧化物歧化酶（MnSOD）在细胞中通过分解线粒体内的超氧化物来抵抗氧化应激中起着至关重要的作用，在病理条件下被硝化和失活。在这项研究中，MnSOD 被证明能催化 PN 介导的自身硝化。PN 衰减和硝基酪氨酸形成的直接光谱观察（k(cat)=9.3×10(2) M(-1) s(-1)）表明，该机制涉及 Mn(2+)和 Mn(3+)之间的氧化还原循环，类似于观察到的超氧化物。通过 MS/MS 分析 MnSOD 胰蛋白酶消化肽，揭示了各种试剂对 MnSOD 内酪氨酸硝化的独特模式，并对其进行了量化。这些分析表明，MnSOD 的七个酪氨酸残基中有三个（Tyr34、Tyr9 和 Tyr11）最容易硝化，并且这些残基的硝化量因硝化剂的性质而异。值得注意的是，PN 介导的硝化，无论是在有 CO2 还是没有 CO2 的情况下，都会导致活性位点酪氨酸 Tyr34 的硝化，而自由扩散的二氧化氮导致 Tyr9 和 Tyr11 的表面硝化。PN-CO2 硝化 Tyr34 的通量分析表明，硝化速率与 PN 与 CO2 的反应动力学一致。这些动力学和 CO2 存在下酪氨酸硝化效率提高 20 倍表明，碳酸盐自由基阴离子（•CO3(-)）在 PN 介导的 MnSOD 硝化中具有特定作用。我们还观察到 Tyr34 的硝化导致酶失活，而 Tyr9 和 Tyr11 的硝化不干扰超氧化物歧化酶活性。Tyr34 硝化导致 MnSOD 活性丧失，这意味着体内的责任试剂是过氧亚硝酸盐，它可以直接或通过•CO3(-)的作用发挥作用。
