Nickel superoxide dismutase: structural and functional roles of His1 and its H-bonding network.
Nickel superoxide dismutase: structural and functional roles of His1 and its H-bonding network.
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DOI：
10.1021/bi501258u
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发表时间：
2015-02-03
期刊：
影响因子：
2.9
通讯作者：
Maroney, Michael J.
中科院分区：
文献类型：
--
作者：
Ryan, Kelly C.;Guce, Abigail I.;Johnson, Olivia E.;Brunold, Thomas C.;Cabelli, Diane E.;Garman, Scott C.;Maroney, Michael J.
镍依赖的超氧化物歧化酶(NiSOD)的晶体结构表明，在His1顶端咪唑配体的N-H和来自六聚体酶相邻亚基的Glu17羧酸盐之间存在一个氢键网络。Glu17和Arg47之间的另一个亚基内氢键支持这种相互作用。在这项研究中，产生了四个突变的NiSOD蛋白来实验评估这个氢键网络的作用，并将结果与DFT计算的先前预测进行比较。对完全缺乏顶端配体的H1a-NiSOD进行了结晶学表征，发现在没有Glu17-His1氢键的情况下，活性中心是无序的。随后的X射线吸收光谱(XAS)表征表明，Ni(II)结合在预期的N2S2平面配位上。尽管存在这些结构上的扰动，H1a-NiSOD突变体仍然是一种活性催化剂，其活性是WT-NiSOD的4%。另外三个突变旨在保留顶端的咪唑配体，但扰乱了氢键网络：R47A-NiSOD，缺乏分子内氢键相互作用；E17R/R47A-NiSOD，保留分子内氢键，但缺乏分子间的Glu17-His1氢键；以及E17A/R47A-NiSOD，缺乏两个氢键相互作用。这些变体通过一系列技术进行了表征，包括镍中心结构的XAS表征，利用脉冲辐射分解产生超氧化物的动力学研究，以及氧化还原活性的EPR和化学探针研究。结果表明，除了计算模型所建议的氧化还原调节作用外，Glu17-His1氢键在镍钩基序的形成中也起着重要的结构作用，镍钩基序是活性中心的关键特征。
Crystal structures of nickel-dependent superoxide dismutases (NiSODs) reveal the presence of a H-bonding network formed between the N-H of the apical imidazole ligand from His1 and the Glu17 carboxylate from a neighboring subunit in the hexameric enzyme. This interaction is supported by another intra-subunit H-bond between Glu17 and Arg47. In this study, four mutant NiSOD proteins were produced to experimentally evaluate the roles of this H-bonding network, and compare the results with prior predictions from DFT calculations. H1A-NiSOD, which lacks the apical ligand entirely, was crystallographically characterized and reveals that in the absence of the Glu17-His1 H-bond, the active site is disordered. Subsequent characterization using X-ray absorption spectroscopy (XAS) shows that Ni(II) is bound in the expected N2S2 planar coordination site. Despite these structural perturbations, the H1A-NiSOD variant is an active catalyst with 4% of WT-NiSOD activity. Three other mutations were designed to preserve the apical imidazole ligand, but perturb the H-bonding network: R47A-NiSOD, lacks the intra-molecular H-bonding interaction, E17R/R47A-NiSOD, which retains the intra-molecular H-bond, but lacks the inter-molecular Glu17-His1 H-bond, and E17A/R47A-NiSOD, which lacks both H-bonding interactions. These variants were characterized by a combination of techniques including XAS characterization of the nickel site structure, kinetic studies employing pulse-radiolytic production of superoxide, and EPR and chemical probes of the redox activity. The results indicate that in addition to the roles in redox tuning suggested by the computational models, the Glu17-His1 H-bond plays an important structural role in the formation of the Ni-hook motif that is a critical feature of the active site.
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影响因子：
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作者：
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影响因子：
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作者：
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作者：
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K