NITRIC-OXIDE SYNTHASE FORMS N-NO-PTERIN AND S-NO-CYS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 诱导型一氧化氮合酶(INOS)产生生物应激水平的一氧化氮(NO)，作为细胞毒性或信号转导的有效介质。然而，这种亚硝化应激对体内iNOS功能的影响还知之甚少。在这里，我们定义了两个特定的非血红素iNOS亚硝化位点，通过结合紫外可见光谱、化学发光、质谱学和X射线结晶学发现。我们用化学发光法检测了酶的周转过程中的自动S亚硝化反应。连接二聚体界面的硫化锌(4)位的选择性S亚硝化促进了二聚体从不稳定的有序到无序的转变。亚硝化的iNOS晶体结构显示，蝶呤辅助因子发生了意外的N-NO修饰。此外，结构上定义的N-NO部分是暴露在溶剂中的，并且可以将NO转移到伴侣。我们研究了谷胱甘肽作为一个潜在的转亚硝化伙伴，因为细胞内的谷胱甘肽浓度很高，而且一氧化氮合酶能形成S-亚硝基谷胱甘肽。我们的计算结果预测了与N-NO-蝶呤相邻的GSH结合部位。此外，我们还用饱和传递差分核磁共振波谱检测了GSH与iNOS的结合。总而言之，这些观察结果解决了先前关于一氧化氮合酶中这种不常见的蝶呤辅助因子的矛盾，并提出了通过N-一氧化氮蝶呤和S-一氧化氮半胱氨酸修饰来调节诱导型一氧化氮合酶活性的方法。这里描述的iNOS自身亚硝化似乎适合于帮助控制NO的产生，以响应细胞条件。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Inducible nitric-oxide synthase (iNOS) produces biologically stressful levels of nitric oxide (NO) as a potent mediator of cellular cytotoxicity or signaling. Yet, how this nitrosative stress affects iNOS function in vivo is poorly understood. Here we define two specific non-heme iNOS nitrosation sites discovered by combining UV-visible spectroscopy, chemiluminescence, mass spectrometry, and x-ray crystallography. We detected auto-S-nitrosylation during enzymatic turnover by using chemiluminescence. Selective S-nitrosylation of the ZnS(4) site, which bridges the dimer interface, promoted a dimer-destabilizing order-to-disorder transition. The nitrosated iNOS crystal structure revealed an unexpected N-NO modification on the pterin cofactor. Furthermore, the structurally defined N-NO moiety is solvent-exposed and available to transfer NO to a partner. We investigated glutathione (GSH) as a potential transnitrosation partner because the intracellular GSH concentration is high and NOS can form S-nitrosoglutathione. Our computational results predicted a GSH binding site adjacent to the N-NO-pterin. Moreover, we detected GSH binding to iNOS with saturation transfer difference NMR spectroscopy. Collectively, these observations resolve previous paradoxes regarding this uncommon pterin cofactor in NOS and suggest means for regulating iNOS activity via N-NO-pterin and S-NO-Cys modifications. The iNOS self-nitrosation characterized here appears appropriate to help control NO production in response to cellular conditions.