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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子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传导的有效介质。然而，这种亚硝化应激如何影响体内iNOS功能还知之甚少。在这里，我们定义了两个特定的非血红素iNOS亚硝化位点发现结合紫外可见光谱，化学发光，质谱，和X射线晶体学。我们检测自动S-亚硝基化酶周转过程中使用化学发光。ZnS（4）位点的选择性S-亚硝基化，其桥接二聚体界面，促进二聚体不稳定的有序到无序转变。亚硝化的iNOS晶体结构揭示了蝶呤辅因子上意想不到的N-NO修饰。此外，结构上限定的N-NO部分是溶剂暴露的，并且可用于将NO转移到配偶体。我们研究了谷胱甘肽（GSH）作为一个潜在的transnitrosation合作伙伴，因为细胞内GSH浓度高，NOS可以形成S-亚硝基谷胱甘肽。我们的计算结果预测GSH结合位点邻近的N-NO-蝶呤。此外，我们用饱和转移差谱检测GSH与iNOS的结合。总的来说，这些观察结果解决了以前关于NOS中这种不常见的蝶呤辅因子的矛盾，并提出了通过N-NO-蝶呤和S-NO-Cys修饰调节iNOS活性的方法。在此描述的iNOS自我亚硝化似乎适合于帮助控制响应于细胞条件的NO产生。

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.