A De Novo Designed 2[4Fe-4S] Ferredoxin Mimic Mediates Electron Transfer
A De Novo Designed 2[4Fe-4S] Ferredoxin Mimic Mediates Electron Transfer
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DOI：
10.1021/ja510621e
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发表时间：
2014-12-20
影响因子：
15
通讯作者：
Ghirlanda, Giovanna
中科院分区：
文献类型：
--
作者：
Roy, Anindya;Sommer, Dayn Joseph;Ghirlanda, Giovanna
[Fe-S]团簇是自然界的模块化电子转移单元，通常排列在支持远程电子转移的链中。尽管有相当大的兴趣，仿生人工系统的设计模仿多簇结合蛋白质，最终目标是将它们整合到人造氧化还原酶，仍然难以捉摸。在这里，我们报告了一种新的双-[4Fe-4S]簇结合蛋白，DSD-Fdm，其中两个簇位于12 A的距离内，与有效的电子转移所需的电子耦合兼容。该设计利用了卷曲螺旋的结构重复以及起始支架（同源二聚体螺旋蛋白（DSD））的对称性。总的来说，DSD核心中的8个疏水残基被8个半胱氨酸残基取代，作为[4Fe-4S]簇的配体。两个[4Fe-4S]簇合物的结合以高产率进行。两个[4Fe-4S]簇位于螺旋束的疏水核心，其特征在于通过各种生物物理技术。载脂蛋白和全蛋白的二级结构是保守的;此外，簇的掺入导致蛋白质相对于化学变性的稳定。最重要的是，这种从头设计的蛋白质可以模仿天然铁氧还蛋白的功能：我们在这里显示，减少的DSD-FDM转移电子到细胞色素c，从而产生减少的细胞色素c化学计量。
[Fe-S] clusters, nature's modular electron transfer units, are often arranged in chains that support long-range electron transfer. Despite considerable interest, the design of biomimetic artificial systems emulating multicluster-binding proteins, with the final goal of integrating them in man-made oxidoreductases, remains elusive. Here, we report a novel bis-[4Fe-4S] cluster binding protein, DSD-Fdm, in which the two clusters are positioned within a distance of 12 A, compatible with the electronic coupling necessary for efficient electron transfer. The design exploits the structural repeat of coiled coils as well as the symmetry of the starting scaffold, a homodimeric helical protein (DSD). In total, eight hydrophobic residues in the core of DSD were replaced by eight cysteine residues that serve as ligands to the [4Fe-4S] clusters. Incorporation of two [4Fe-4S] clusters proceeds with high yield. The two [4Fe-4S] clusters are located in the hydrophobic core of the helical bundle as characterized by various biophysical techniques. The secondary structure of the apo and holo proteins is conserved; further, the incorporation of clusters results in stabilization of the protein with respect to chemical denaturation. Most importantly, this de novo designed protein can mimic the function of natural ferredoxins: we show here that reduced DSD-Fdm transfers electrons to cytochrome c, thus generating the reduced cyt c stoichiometrically.