Electrochemistry of the [4Fe4S] Cluster in Base Excision Repair Proteins: Tuning the Redox Potential with DNA.
Electrochemistry of the [4Fe4S] Cluster in Base Excision Repair Proteins: Tuning the Redox Potential with DNA.
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DOI：
10.1021/acs.langmuir.6b04581
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发表时间：
2017-03-14
期刊：
影响因子：
--
通讯作者：
Barton JK
中科院分区：
文献类型：
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作者：
Bartels PL;Zhou A;Arnold AR;Nuñez NN;Crespilho FN;David SS;Barton JK
大肠杆菌内切酶III（EndoIII）和MutY是含有[4Fe 4S]簇的DNA糖基化酶，其用于在氧化应激后维持基因组的完整性。高取向热解石墨（HOPG）的电化学研究表明，DNA结合EndoIII导致一个大的负移中点电位的集群，与稳定的氧化[4Fe 4S]3+的形式。然而，光滑、疏水的HOPG表面对于在没有DNA的情况下与蛋白质一起工作是不理想的。在这项工作中，我们使用热解石墨边缘电极上的薄膜伏安法来克服这些限制。在不存在DNA的情况下，改进的吸附导致EndoIII和MutY的大量信号，并且在存在DNA的情况下保留大的负电位偏移。相比之下，EndoIII突变体E200 K，Y205 H和K208 E，这提供了在簇附近的静电扰动，都显示出野生型的误差内的无DNA电位;类似地，带负电荷的聚-L谷氨酸的存在不会导致显着的电位偏移。总的来说，结合到DNA聚阴离子是调节[4Fe 4S]簇的氧化还原电位的主导作用，这有助于解释为什么迄今为止研究的所有具有[4Fe 4S]簇的DNA结合蛋白具有相似的DNA结合电位。
Escherichia coli Endonuclease III (EndoIII) and MutY are DNA glycosylases that contain [4Fe4S] clusters and that serve to maintain the integrity of the genome after oxidative stress. Electrochemical studies on highly-oriented pyrolytic graphite (HOPG) revealed that DNA binding by EndoIII leads to a large negative shift in midpoint potential of the cluster, consistent with stabilization of the oxidized [4Fe4S]3+ form. However, the smooth, hydrophobic HOPG surface is non-ideal for working with proteins in the absence of DNA. In this work, we use thin film voltammetry on a pyrolytic graphite edge electrode to overcome these limitations. Improved adsorption leads to substantial signals for both EndoIII and MutY in the absence of DNA, and a large negative potential shift is retained with DNA present. In contrast, the EndoIII mutants E200K, Y205H, and K208E, which provide electrostatic perturbations in the vicinity of the cluster, all show DNA-free potentials within error of wild type; similarly, the presence of negatively charged poly-L glutamate does not lead to a significant potential shift. Overall, binding to the DNA polyanion is the dominant effect in tuning the redox potential of the [4Fe4S] cluster, helping to explain why all DNA-binding proteins with [4Fe4S] clusters studied to date have similar DNA-bound potentials.