Cheng Zishuo, Landry Aaron P, Wang Yiming, Ding Huangen
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803.
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803.
J Biol Chem. 2017 Feb 24;292(8):3146-3153. doi: 10.1074/jbc.M116.766774. Epub 2017 Jan 12.
Iron-sulfur proteins are among the primary targets of nitric oxide in cells. Previous studies have shown that iron-sulfur clusters hosted by cysteine residues in proteins are readily disrupted by nitric oxide forming a protein-bound dinitrosyl iron complex, thiolate-bridged di-iron tetranitrosyl complex, or octanitrosyl cluster. Here we report that human mitochondrial protein Miner2 [2Fe-2S] clusters can bind nitric oxide without disruption of the clusters. Miner2 is a member of a new CDGSH iron-sulfur protein family that also includes two mitochondrial proteins: the type II diabetes-related mitoNEET and the Wolfram syndrome 2-linked Miner1. Miner2 contains two CDGSH motifs, and each CDGSH motif hosts a [2Fe-2S] cluster via three cysteine and one histidine residues. Binding of nitric oxide in the reduced Miner2 [2Fe-2S] clusters produces a major absorption peak at 422 nm without releasing iron or sulfide from the clusters. The EPR measurements and mass spectrometry analyses further reveal that nitric oxide binds to the reduced [2Fe-2S] clusters in Miner2, with each cluster binding one nitric oxide. Although the [2Fe-2S] cluster in purified human mitoNEET and Miner1 fails to bind nitric oxide, a single mutation of Asp-96 to Val in mitoNEET or Asp-123 to Val in Miner1 facilitates nitric oxide binding in the [2Fe-2S] cluster, indicating that a subtle change of protein structure may switch mitoNEET and Miner1 to bind nitric oxide. The results suggest that binding of nitric oxide in the CDGSH-type [2Fe-2S] clusters in mitochondrial protein Miner2 may represent a new nitric oxide signaling mode in cells.
铁硫蛋白是细胞中一氧化氮的主要作用靶点之一。先前的研究表明，蛋白质中由半胱氨酸残基承载的铁硫簇很容易被一氧化氮破坏，形成蛋白质结合的二亚硝酰铁络合物、硫醇盐桥连的二铁四亚硝酰络合物或八亚硝酰簇。在此，我们报告人类线粒体蛋白Miner2的[2Fe-2S]簇能够结合一氧化氮而不破坏该簇。Miner2是一个新的CDGSH铁硫蛋白家族的成员，该家族还包括两种线粒体蛋白：与II型糖尿病相关的mitoNEET和与沃夫勒姆综合征2相关的Miner1。Miner2包含两个CDGSH基序，每个CDGSH基序通过三个半胱氨酸和一个组氨酸残基承载一个[2Fe-2S]簇。一氧化氮与还原态的Miner2 [2Fe-2S]簇结合会在422 nm处产生一个主要吸收峰，且不会从簇中释放铁或硫。电子顺磁共振测量和质谱分析进一步表明，一氧化氮与Miner2中还原态的[2Fe-2S]簇结合，每个簇结合一个一氧化氮。尽管纯化的人类mitoNEET和Miner1中的[2Fe-2S]簇不能结合一氧化氮，但mitoNEET中的Asp-96突变为Val或Miner1中的Asp-123突变为Val会促进一氧化氮与[2Fe-2S]簇的结合，这表明蛋白质结构的细微变化可能会使mitoNEET和Miner1转变为能够结合一氧化氮。这些结果表明，线粒体蛋白Miner2中CDGSH型[2Fe-2S]簇与一氧化氮的结合可能代表了细胞中一种新的一氧化氮信号传导模式。
