Nitrosation by peroxynitrite: use of phenol as a probe.
Nitrosation by peroxynitrite: use of phenol as a probe.
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DOI：
10.1006/abbi.1998.0825
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发表时间：
1998-10
影响因子：
3.9
通讯作者：
R. Uppu;J. Lemercier;G. Squadrito;H. Zhang;R. Bolzan;W. Pryor
中科院分区：
文献类型：
--
作者：
R. Uppu;J. Lemercier;G. Squadrito;H. Zhang;R. Bolzan;W. Pryor
亚硝化是一氧化氮代谢的重要途径，产生S-亚硝基硫醇，可能是关键的信号转导物种。长期以来，已知过氧亚硝酸盐与芳族化合物在pH 5至8范围内的反应产生羟基化和硝化产物。然而，我们在这里提出的证据表明，过氧亚硝酸盐也可以促进亲核试剂的亚硝化。我们选择苯酚作为底物，因为亚硝化反应是在研究过氧化亚硝酸根对苯酚的羟基化和硝化模式的CO2调节期间首次认识到的（Lemercier等人，《生物化学与生物物理学基础》345，160-170，1997）。在pH 7.0时检测到主要亚硝化产物4-亚硝基苯酚，沿着2-和4-硝基苯酚;在pH >/= 8.0时，4-亚硝基苯酚成为主要产物。4-亚硝基苯酚的产率甚至在pH 11.1、1.比苯酚的pKa高2个单位，表明参与反应的是酚盐离子，而不是苯酚。过氧化氢不是作为副产物形成的。亚硝化反应在苯酚中为零级，在过氧亚硝酸盐中为一级，这表明酚盐离子与来自过氧亚硝酸盐的活化亚硝化物质反应，而不是与过氧亚硝酸盐本身反应。在最佳条件下，4-亚硝基苯酚的产率与2-和4-硝基苯酚的产率相当，表明亚硝化反应与酚类化合物被过氧亚硝酸根硝化一样重要。低浓度的CO2有利于亚硝化反应，但过量的CO2会显著降低4-亚硝基苯酚的产率。如果O=N-OO-与过氧亚硝酸根阴离子-CO2加合物（O=N-OOCO-2）或由其衍生的次级中间体（包括硝基碳酸根阴离子（O2 N-OCO-2）、碳酸根自由基（CO*-3）和 *NO2）反应，则CO2的双重效应可以合理化。从这些反应中得到的产物可以被设想为活化的中间体X-N=O（其中X是-OONO 2、-NO2或-CO-3），其可以将亚硝酰基阳离子（NO+）转移到酚盐离子。苯酚亚硝化的另一种机制是酚盐离子被CO*-3单电子氧化产生苯氧基自由基，O=N-OO-被CO*-3氧化产生亚硝酰二氧自由基（O=N-OO*），亚硝酰二氧自由基分解产生 *NO和O2;*NO然后与苯氧基自由基反应产生亚硝基苯酚。这两种机制是一致的NO-2和O2的高产率在过氧亚硝酸盐的碱性分解和N-3对苯酚的亚硝化过氧亚硝酸盐和过氧亚硝酸盐/CO2加合物的有效抑制作用。讨论了过氧亚硝酸盐介导的亚硝化反应的生物学意义。
Nitrosation is an important pathway in the metabolism of nitric oxide, producing S-nitrosothiols that may be critical signal transduction species. The reaction of peroxynitrite with aromatic compounds in the pH range of 5 to 8 has long been known to produce hydroxylated and nitrated products. However, we here present evidence that peroxynitrite also can promote the nitrosation of nucleophiles. We chose phenol as a substrate because the nitrosation reaction was first recognized during a study of the CO2-modulation of the patterns of hydroxylation and nitration of phenol by peroxynitrite (Lemercier et al., Arch. Biochem. Biophys. 345, 160-170, 1997). 4-Nitrosophenol, the principal nitrosation product, is detected at pH 7.0, along with 2- and 4-nitrophenols; 4-nitrosophenol becomes the dominant product at pH >/= 8.0. The yield of 4-nitrosophenol continues to increase even after pH 11.1, 1. 2 units above the pKa of phenol, suggesting that the phenolate ion, and not phenol, is involved in the reaction. Hydrogen peroxide is not formed as a by-product. The nitrosation reaction is zero-order in phenol and first-order in peroxynitrite, suggesting the phenolate ion reacts with an activated nitrosating species derived from peroxynitrite, and not with peroxynitrite itself. Under optimal conditions, the yields of 4-nitrosophenol are comparable to those of 2- and 4-nitrophenols, indicating that the nitrosation reaction is as significant as the nitration of phenolic compounds by peroxynitrite. Low concentrations of CO2 facilitate the nitrosation reaction, but excess CO2 dramatically reduces the yield of 4-nitrosophenol. The dual effects of CO2 can be rationalized if O=N-OO- reacts with the peroxynitrite anion-CO2 adduct (O=N-OOCO-2) or secondary intermediates derived from it, including the nitrocarbonate anion (O2N-OCO-2), the carbonate radical (CO*-3), and *NO2. The product resulting from these reactions can be envisioned as an activated intermediate X-N=O (where X is -OONO2, -NO2, or -CO-3) that could transfer a nitrosyl cation (NO+) to the phenolate ion. An alternative mechanism for the nitrosation of phenol involves the one-electron oxidation of the phenolate ion by CO*-3 to give the phenoxyl radical and the oxidation of O=N-OO- by CO*-3 to give a nitrosyldioxyl radical (O=N-OO*), which decomposes to give *NO and O2; the *NO then reacts with the phenoxyl radical giving nitrosophenol. Both mechanisms are consistent with the high yields of NO-2 and O2 during the alkaline decomposition of peroxynitrite and the potent inhibitory effect of N-3 on the nitrosation of phenol by peroxynitrite and peroxynitrite/CO2 adducts. The biological significance of the peroxynitrite-mediated nitrosations is discussed.