Iron Insertion at the Assembly Site of the ISCU Scaffold Protein Is a Conserved Process Initiating Fe-S Cluster Biosynthesis.
Iron Insertion at the Assembly Site of the ISCU Scaffold Protein Is a Conserved Process Initiating Fe-S Cluster Biosynthesis.
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DOI：
10.1021/jacs.2c06338
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发表时间：
2022-09-28
影响因子：
15
通讯作者：
D'Autreaux, Benoit
中科院分区：
文献类型：
--
作者：
Srour, Batoul;Gervason, Sylvain;Hoock, Maren Hellen;Monfort, Beata;Want, Kristian;Larkem, Djabir;Trabelsi, Nadine;Landrot, Gautier;Zitolo, Andrea;Fonda, Emiliano;Etienne, Emilien;Gerbaud, Guillaume;Mueller, Christina Sophia;Oltmanns, Jonathan;Gordon, Jesse B.;Yadav, Vishal;Kleczewska, Malgorzata;Jelen, Marcin;Toledano, Michel B.;Dutkiewicz, Rafal;Goldberg, David P.;Schuenemann, Volker;Guigliarelli, Bruno;Burlat, Benedicte;Sizun, Christina;D'Autreaux, Benoit
铁硫（Fe-S）团簇是由高度保守的多蛋白机制在支架蛋白上合成的蛋白质假体。Fe-S簇在ISCU支架蛋白中的生物合成是由亚铁插入和硫获取启动的，其机制尚不明确。值得注意的是，尚不清楚铁最初是通过N/O配体结合到ISCU富含半胱氨酸的组装位点还是通过无半胱氨酸的辅助位点。我们在这里通过SEC， CD和Mössbauer光谱显示，铁通过一个或两个半胱氨酸结合到原核生物和真核生物的单体ISCU蛋白的组装位点，称为1-Cys和2-Cys形式。后者在pH 8.0时占主导地位，并与Fe-S簇组装活性相关，而前者在更酸性的pH下增加，并伴有游离铁，表明它是铁插入过程的中间产物。不结合组装位点的铁非特异性结合到聚合的ISCU上，排除了ISCU中存在结构上确定的辅助位点。通过位点定向诱变、CD、NMR、XAS、Mössbauer和EPR光谱对2-Cys结构进行了表征，结果表明，铁中心与组装位点的4个严格保守的氨基酸（Cys35、Asp37、Cys61和His103）配位成四面体结构。硫受体Cys104在非常近的距离上被发现，当His103缺失时，它显然与铁中心结合，这可能使铁依赖性硫获得。综上所述，这些数据为阐明Fe-S簇组装过程提供了结构基础，并确立了在ISCU组装位点插入一个亚铁引发Fe-S簇生物合成是一个保守的机制。
Iron-sulfur (Fe-S) clusters are prosthetic groups of proteins biosynthesized on scaffold proteins by highly conserved multi-protein machineries. Fe-S clusters biosynthesis into the ISCU scaffold protein is initiated by ferrous iron insertion followed by sulfur acquisition, via a still elusive mechanism. Notably, whether iron initially binds to the ISCU cysteine-rich assembly site or to a cysteine-less auxiliary site via N/O ligands remains unclear. We show here by SEC, CD and Mössbauer spectroscopies that iron binds to the assembly site of monomeric ISCU proteins from prokaryotes and eukaryotes via either one or two cysteines, referred to the 1-Cys and 2-Cys forms. The latter predominated at pH 8.0 and correlated with Fe-S cluster assembly activity, whereas the former increased at a more acidic pH, together with free iron, suggesting that it constitutes an intermediate of the iron insertion process. Iron not binding to the assembly site was non-specifically bound to aggregated ISCU, ruling out the existence of a structurally defined auxiliary site in ISCU. Characterization of the 2-Cys form by site-directed mutagenesis, CD, NMR, XAS, Mössbauer and EPR spectroscopies showed that the iron center is coordinated by four strictly conserved amino acids of the assembly site: Cys35, Asp37, Cys61 and His103 in a tetrahedral geometry. The sulfur receptor Cys104 was found at very close distance and apparently bound to the iron center when His103 was missing, which may enable iron-dependent sulfur acquisition. Altogether, these data provide the structural basis to elucidate the Fe-S cluster assembly process and establish that the initiation of Fe-S cluster biosynthesis by insertion of a ferrous iron in the assembly site of ISCU is a conserved mechanism.
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