STRUCTURE-FUNCTION STUDIES OF [2FE-2S] FERREDOXINS
STRUCTURE-FUNCTION STUDIES OF [2FE-2S] FERREDOXINS
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DOI：
10.1007/bf00763220
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发表时间：
1994-02-01
影响因子：
3
通讯作者：
MARKLEY, JL
中科院分区：
文献类型：
--
作者：
HOLDEN, HM;JACOBSON, BL;MARKLEY, JL
在大肠杆菌中过表达[2Fe-2S]铁氧化还原蛋白的能力开辟了令人兴奋的研究机会。高分辨率的X-射线结构已被确定为野生型铁氧还蛋白产生的营养和异形体形式的鱼腥藻菌株7120（在其氧化状态），这些已被比较的结构信息来自多维，多核NMR光谱。在这些蛋白质在其氧化和还原状态的电子离域已被研究的H-1，H-2，C-13，和N-15 NMR光谱。定点诱变已用于制备这些铁氧化还原蛋白的变体。突变体（超过50个）的营养铁氧还蛋白已被设计成探讨集群组装和稳定的问题，并确定哪些残基是重要的识别和电子转移到氧化还原伙伴鱼腥藻铁氧还蛋白还原酶。结果表明，丝氨酸可以取代半胱氨酸在每个四个集群连接位点，仍然支持集群组装。电子转移已被证明与四个突变体中的三个。虽然这些突变体比野生型铁氧还蛋白更不稳定，但已经可以确定其中一个（C49 S）的X射线结构，并通过EPR和NMR表征所有四个突变体。突变已经鉴定了营养铁氧还蛋白的残基65和94对于与还原酶的相互作用至关重要。已经通过X射线衍射分析获得了几种额外突变体的三维模型：T48 S、A50 V、E94 K（在功能测定中活性比野生型低四个数量级）和A43 S/A45 S/T48 S/A50 N（四重突变体）。
The ability to overexpress [2Fe-2S] ferredoxins in Escherichia coli has opened up exciting research opportunities. High-resolution x-ray structures have been determined for the wild-type ferredoxins produced by the vegetative and heterocyst forms of Anabaena strain 7120 (in their oxidized states), and these have been compared to structural information derived from multidimensional, multinuclear NMR spectroscopy. The electron delocalization in these proteins in their oxidized and reduced states has been studied by H-1, H-2, C-13, and N-15 NMR spectroscopy. Site-directed mutagenesis has been used to prepare variants of these ferredoxins. Mutants (over 50) of the vegetative ferredoxin have been designed to explore questions about cluster assembly and stabilization and to determine which residues are important for recognition and electron transfer to the redox partner Anabaena ferredoxin reductase. The results have shown that serine can replace cysteine at each of the four cluster attachment sites and still support cluster assembly. Electron transfer has been demonstrated with three of the four mutants. Although these mutants are less stable than the wild-type ferredoxin, it has been possible to determine the x-ray structure of one (C49S) and to characterize all four by EPR and NMR. Mutagenesis has identified residues 65 and 94 of the vegetative ferredoxin as crucial to interaction with the reductase, Three-dimensional models have been obtained by x-ray diffraction analysis for several additional mutants: T48S, A50V, E94K (four orders of magnitude less active than wild type in functional assays), and A43S/A45S/T48S/A50N (quadruple mutant).