Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins
Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins
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DOI：
10.1074/jbc.m111.228726
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发表时间：
2011-08-05
影响因子：
4.8
通讯作者：
Rouhier, Nicolas
中科院分区：
文献类型：
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作者：
Couturier, Jeremy;Stroeher, Elke;Rouhier, Nicolas
与硫氧还蛋白不同，谷氧还蛋白参与铁-硫簇组装和特定二硫化物（即蛋白质-谷胱甘肽加合物）的还原，因此它们也是叶绿体代谢的重要氧化还原调节剂。使用 GFP 融合，AtGrxC5 同种型（仅存在于十字花科植物中）被证明位于叶绿体中。拟南芥 GrxC5（WCSYC 活性位点）与杨树 GrxS12（WCSYS 活性位点）（一种叶绿体旁系同源物）的生化、结构和光谱特性的比较表明，与仅无辅基单体的 GrxS12 亚型相反，AtGrxC5 在大肠杆菌中表达时以两种形式存在。单体脱辅基蛋白具有介导质体甲硫氨酸亚砜还原酶 B1 和过氧化还原蛋白 IIE 回收的去谷胱甘肽活性，而二聚全蛋白则包含 [2Fe-2S] 簇。定点诱变实验和 AtGrxC5 全息体 X 射线晶体结构的解析表明，尽管不参与其连接，但簇形成需要第二个活性位点半胱氨酸 (Cys(32)) 的存在。此外，硫醇滴定、荧光测量和质谱分析表明，尽管存在二硫醇活性位点，AtGrxC5 不会形成任何分子间或分子内二硫键，并且其活性完全依赖于单硫醇机制。
Unlike thioredoxins, glutaredoxins are involved in iron-sulfur cluster assembly and in reduction of specific disulfides (i.e. protein-glutathione adducts), and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, was shown to be localized in chloroplasts. A comparison of the biochemical, structural, and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicated that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the x-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys(32)) is required for cluster formation. In addition, thiol titrations, fluorescence measurements, and mass spectrometry analyses showed that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter-or intramolecular disulfide bond and that its activity exclusively relies on a monothiol mechanism.