Mass Spectrometric Identification of [4Fe-4S](NO)x Intermediates of Nitric Oxide Sensing by Regulatory Iron-Sulfur Cluster Proteins
Mass Spectrometric Identification of [4Fe-4S](NO)x Intermediates of Nitric Oxide Sensing by Regulatory Iron-Sulfur Cluster Proteins
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DOI：
10.1002/chem.201806113
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发表时间：
2019-03-07
影响因子：
4.3
通讯作者：
Le Brun, Nick E.
中科院分区：
文献类型：
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作者：
Crack, Jason C.;Le Brun, Nick E.
一氧化氮（NO）既可以作为细胞毒素，也可以作为信号分子。在这两种情况下，与铁硫（Fe-S）簇蛋白的反应起着重要作用，因为Fe-S簇对NO起反应，因此是一般NO诱导损伤（毒性）的主要部位。这种对亚硝基化的敏感性在越来越多的调节蛋白中被利用，这些调节蛋白通过Fe-S簇感应NO。虽然关于团簇亚硝基化产物的信息正在出现，但中间体的检测和鉴定仍然是一个主要挑战，因为它们的瞬态性质和难以区分光谱相似的铁no物种。本文作者利用非变性质谱法对NO传感的Fe-S簇调控因子NsrR和WhiD进行了研究，其中蛋白质与Fe/S/NO之间的非共价相互作用得以保留。这些数据为亚硝基化反应提供了深刻的见解，首次鉴定了蛋白质结合的单、二和四硝基[4Fe-4S]团簇配合物([4Fe-4S](NO), [4Fe-4S](NO)(2)和[4Fe-4S]（NO)(4)）作为产物Roussin红酯（RRE）和Roussin黑盐（RBS）样物质形成途径的中间体。这些数据使NsrR和WhiD的亚硝基化机制得以阐明和明确区分。
Nitric oxide (NO) can function as both a cytotoxin and a signalling molecule. In both cases, reaction with iron-sulfur (Fe-S) cluster proteins plays an important role because Fe-S clusters are reactive towards NO and so are a primary site of general NO-induced damage (toxicity). This sensitivity to nitrosylation is harnessed in the growing group of regulatory proteins that function in sensing of NO via an Fe-S cluster. Although information about the products of cluster nitrosylation is now emerging, detection and identification of intermediates remains a major challenge, due to their transient nature and the difficulty in distinguishing spectroscopically similar iron-NO species. Here we report studies of the NO-sensing Fe-S cluster regulators NsrR and WhiD using non-denaturing mass spectrometry, in which non-covalent interactions between the protein and Fe/S/NO species are preserved. The data provide remarkable insight into the nitrosylation reactions, permitting identification, for the first time, of protein-bound mono-, di- and tetranitrosyl [4Fe-4S] cluster complexes ([4Fe-4S](NO), [4Fe-4S])(NO)(2) and [4Fe-4S](NO)(4)) as intermediates along pathways to formation of product Roussin's red ester (RRE) and Roussin's black salt (RBS)-like species. The data allow the nitrosylation mechanisms of NsrR and WhiD to be elucidated and clearly distinguished.