IscA, an alternate scaffold for Fe-S cluster biosynthesis
IscA, an alternate scaffold for Fe-S cluster biosynthesis
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DOI：
10.1021/bi015656z
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发表时间：
2001-11-20
期刊：
影响因子：
2.9
通讯作者：
Johnson, MK
中科院分区：
文献类型：
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作者：
Krebs, C;Agar, JN;Johnson, MK
在大肠杆菌中表达了棕色固氮菌(Azotobacter Vinelandii)nif调控子中的IscA同源物，命名为(NIF)IscA。纯化的(NIF)ISCA被发现是一个11 kDa亚基的同源二聚体，在其分离的形式中不含金属中心或其他假体基团。通过UV-Vis吸收、穆斯堡尔谱、共振拉曼光谱、变温磁圆二色谱和电子顺磁共振光谱的结合，研究了(Nif)IscA与铁结合和组装Fe-S簇生物合成的能力，并通过UV-Vis吸收、穆斯堡尔谱、共振拉曼光谱、变温磁圆二色谱和EPR光谱相结合的方法，评估了(Nif)IscA在Fe-S簇生物合成中的可能作用。虽然(Nif)IscA被发现在一个以半胱氨酸连接的四面体配位环境中与亚铁离子结合，但低结合亲和力与作为金属六配位试剂将亚铁离子输送到其他Fe-S簇组装蛋白的特定作用相反。相反，(Nif)IscA作为Fe-S簇生物合成的替代支架蛋白的作用是基于NIF指导的每个(Nif)IscA同源二聚体大约一个不稳定的[4Fe-4S](2+)簇的组装，通过瞬时的[2Fe-2S](2+)簇中间体。用紫外-可见吸收光谱和穆斯堡尔谱对簇合物的组装过程进行了时间监测，并用共振拉曼光谱对中间体[2Fe-2S](2+)进行了表征。[2Fe-2S](2+)中心的穆斯堡尔谱和共振拉曼光谱与完全的半胱氨酸基连接一致。在所有ISCA蛋白中都存在三个保守的半胱氨酸残基，并且观察到每个同源二聚体大约有一个[2Fe-2S](2+)或一个[4Fe-4S](2+)的簇化学计量比表明，这两种簇类型都是亚基桥联。此外，(NIF)ISCA通过亚铁离子还原硫磺，实现了铁和硫的耦合输送。铁-S支架蛋白偶联这两种有毒和活性的铁-S簇前体物的能力对于最大限度地降低细胞内游离亚铁离子和硫离子的浓度可能是重要的。在光谱和分析结果的基础上，提出了在(NIF)ISCA上进行NIFS定向团簇组装的机制方案。有人认为，IscA家族蛋白为NifU和IscU蛋白提供了替代的支架，以介导Nif特异性和一般性的Fe-S簇组装。
An IscA homologue within the nif regulon of Azotobacter vinelandii, designated (Nif)IscA, was expressed in Escherichia coli and purified to homogeneity. Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. Possible roles for (Nif)IscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, Mossbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies, Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. Rather, a role for (Nif)IscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S](2+) cluster per (Nif)IscA homodimer, via a transient [2Fe-2S](2+) cluster intermediate. The cluster assembly process was monitored temporally using UV-vis absorption and Mossbauer spectroscopy, and the intermediate [2Fe-2S](2+)-containing species was additionally characterized by resonance Raman spectroscopy. The Mossbauer and resonance Raman properties of the [2Fe-2S](2+) center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S](2+) or one [4Fe-4S](2+) per homodimer suggest that both cluster types are subunit bridging. In addition, (Nif)IscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on (Nif)IscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.