Arabidopsis thaliana Nfu2 Accommodates [2Fe-2S] or [4Fe-4S] Clusters and Is Competent for in Vitro Maturation of Chloroplast [2Fe-2S] and [4Fe-4S] Cluster-Containing Proteins
Arabidopsis thaliana Nfu2 Accommodates [2Fe-2S] or [4Fe-4S] Clusters and Is Competent for in Vitro Maturation of Chloroplast [2Fe-2S] and [4Fe-4S] Cluster-Containing Proteins
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DOI：
10.1021/bi4007622
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发表时间：
2013-09-24
期刊：
影响因子：
2.9
通讯作者：
Johnson, Michael K.
中科院分区：
文献类型：
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作者：
Gao, Huanyao;Subramanian, Sowmya;Johnson, Michael K.
在许多生物体中，nfu型蛋白在铁硫（Fe-S）簇的生物发生中是必不可少的。拟南芥（Arabidopsis thaliana）中编码叶绿体特异性nfii型蛋白Nfu2 （AtNfu2）的基因缺失与低水平Fe-S簇结合等许多表型有关。在这里，我们报道重组AtNfu2能够组装[2Fe-2S]和[4Fe-4S]簇。分析数据和凝胶过滤研究支持一个[2Fe-2S]簇/同型四聚体和一个[4Fe-4S]簇/同型二聚体的簇/蛋白质化学计量学。利用紫外可见吸收光谱、圆二色光谱、共振拉曼光谱和穆斯堡尔光谱研究了在Nfu2上组装的团簇的性质、性质和转移。结果与保守的CXXC基序中半胱氨酸协调的亚基桥接[2Fe-2S](2)和[4Fe-4S]（2+）簇一致。研究结果还揭示了Nfu2通过完整、快速和定量的簇转移对叶绿体Fe-S蛋白成熟的特异性。[2Fe-2S]簇结合的Nfu2被证明是glutaredoxin S16有效的[2Fe-2S]2+簇供体，而不是glutaredoxin S14。此外，[4Fe-4S]簇结合的Nfu2被证明是腺苷5'-磷酸硫酸还原酶（APR1）的一个非常快速和有效的[4Fe-4Sr]（+2）簇供体，酵母双杂交研究表明，APR1与Nfia形成复合物，但不与Mil和Nfu3（另外两种叶绿体Nfu蛋白）形成复合物。这种簇转移可能与生理相关，对植物代谢尤其重要，因为APR1催化还原性硫同化的第二步，最终导致半胱氨酸、甲硫氨酸、谷胱甘肽和Fe-S簇的生物合成。
Nfu-type proteins are essential in the biogenesis of iron-sulfur (Fe-S) clusters in numerous organisms. A number of phenotypes including low levels of Fe-S cluster incorporation are associated with the deletion of the gene encoding a chloroplast-specific Nfii-type protein, Nfu2 from Arabidopsis thaliana (AtNfu2). Here, we report that recombinant AtNfu2 is able to assemble both [2Fe-2S] and [4Fe-4S] clusters. Analytical data and gel filtration studies support cluster/protein stoichiometries of one [2Fe-2S] cluster/homotetramer and one [4Fe-4S] cluster/homodimer. The combination of UV visible absorption and circular dichroism and resonance Raman and Mossbauer spectroscopies has been employed to investigate the nature, properties, and transfer of the clusters assembled on Nfu2. The results are consistent with subunit-bridging [2Fe-2S](2) and [4Fe-4S](2+) clusters coordinated by the cysteines in the conserved CXXC motif. The results also provided insight into the specificity of Nfu2 for the maturation of chloroplastic Fe-S proteins via intact, rapid, and quantitative cluster transfer. [2Fe-2S] cluster-bound Nfu2 is shown to be an effective [2Fe-2S]2+ cluster donor for glutaredoxin S16 but not glutaredoxin S14. Moreover, [4Fe-4S] cluster-bound Nfu2 is shown to be a very rapid and efficient [4Fe-4Sr](+2) cluster donor for adenosine 5'-phosphosulfate reductase (APR1), and yeast two-hybrid studies indicate that APR1 forms a complex with Nfia but not with Mil and Nfu3, the two other chloroplastic Nfu proteins. This cluster transfer is likely to be physiologically relevant and is particularly significant for plant metabolism as APR1 catalyzes the second step in reductive sulfur assimilation, which ultimately results in the biosynthesis of cysteine, methionine, glutathione, and Fe-S clusters.