Spectroscopic and functional characterization of the [2Fe-2S] scaffold protein Nfu from Synechocystis PCC6803.
Spectroscopic and functional characterization of the [2Fe-2S] scaffold protein Nfu from Synechocystis PCC6803.
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DOI：
10.1016/j.biochi.2021.09.013
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发表时间：
2022-01
期刊：
影响因子：
3.9
通讯作者：
Cowan JA
中科院分区：
文献类型：
--
作者：
Thompson Z;Fidai I;Wachnowsky C;Hendricks AL;Cowan JA
铁-硫簇是各种重要代谢过程中普遍存在的辅因子。保护其生物合成和运输所需的蛋白质使简单的细菌能够被用作模型，以帮助探索高等生物体中这些复杂的途径。蓝藻是这些过程中被研究最多的生物之一，因为它们是单细胞的，可以在光自养和异养条件下生存。在这里，我们报告了聚球藻PCC6803 NifU(现在命名为SyNfu)作为该生物体中铁-硫簇生物合成所需的主要支架蛋白的潜在作用。SyNfu是一种折叠良好的蛋白质，具有明显的二级结构元素，圆二色谱和1H-15N HSQC核磁共振峰的分散模式证明了这一点，并容易重组为[2Fe-2S]二聚体蛋白质复合体。团簇交换实验表明，谷胱甘肽可以从HALO-SyNfu中提取团簇，但转移是单向的。我们还证实了SyNfu能够将簇转移到人的铁氧还蛋白1和铁氧还蛋白2，同时也证明了将簇转移到单硫醇谷氧还蛋白3和二硫醇谷氧还蛋白2的能力。这一证据支持了这样的假设，即SyNfu确实是集胞藻的主要支架蛋白，因为它被证明是在没有光自养条件下生存所需的唯一蛋白质。与其他NFU型簇供体和其他支架和载体蛋白(如ISCU)类似，DSC表明SyNfu在结构上不如常规蛋白质供体稳定，同时保留了1H-15N HSQC核磁共振实验所代表的相对明确的三级结构。
Iron-sulfur clusters are ubiquitous cofactors required for various essential metabolic processes. Conservation of proteins required for their biosynthesis and trafficking allows for simple bacteria to be used as models to aid in exploring these complex pathways in higher organisms. Cyanobacteria are among the most investigated organisms for these processes, as they are unicellular and can survive under photoautotrophic and heterotrophic conditions. Herein, we report the potential role of Synechocystis PCC6803 NifU (now named SyNfu) as the principal scaffold protein required for iron-sulfur cluster biosynthesis in that organism. SyNfu is a well-folded protein with distinct secondary structural elements, as evidenced by circular dichroism and a well-dispersed pattern of 1H-15N HSQC NMR peaks, and readily reconstitutes as a [2Fe-2S] dimeric protein complex. Cluster exchange experiments show that glutathione can extract the cluster from holo-SyNfu, but the transfer is unidirectional. We also confirm the ability of SyNfu to transfer cluster to both human ferredoxin 1 and ferredoxin 2, while also demonstrating the capacity to deliver cluster to both monothiol glutaredoxin 3 and dithiol glutaredoxin 2. This evidence supports the hypothesis that SyNfu indeed serves as the main scaffold protein in Synechocystis, as it has been shown to be the only protein required for viability in the absence of photoautotrophic conditions. Similar to other NFU-type cluster donors and other scaffold and carrier proteins, such as ISCU, SyNfu is shown by DSC to be structurally less stable than regular protein donors, while retaining a relatively well-defined tertiary structure as represented by 1H-15N HSQC NMR experiments.
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影响因子：
3
作者：
Kim WY;Kang S;Kim BC;Oh J;Cho S;Bhak J;Choi JS
通讯作者：
Choi JS
影响因子：
4.8
作者：
Cai, Kai;Frederick, Ronnie O.;Markley, John L.
通讯作者：
Markley, John L.
影响因子：
4.8
作者：
Mansy, SS;Wu, SP;Cowan, JA
通讯作者：
Cowan, JA
影响因子：
2.9
作者：
Li, Jingwei;Ding, Shu;Cowan, J. A.
通讯作者：
Cowan, J. A.
DOI：
10.1073/pnas.1114372109
发表时间：
2012-01-10
影响因子：
11.1
作者：
Kim, Jin Hae;Tonelli, Marco;Markley, John L.
通讯作者：
Markley, John L.