Ryan Kelly C, Guce Abigail I, Johnson Olivia E, Brunold Thomas C, Cabelli Diane E, Garman Scott C, Maroney Michael J
Department of Chemistry, University of Massachusetts at Amherst , 104 Lederle Graduate Research Tower A, 710 North Pleasant Street, Amherst, Massachusetts 01003, United States.
Biochemistry. 2015 Feb 3;54(4):1016-27. doi: 10.1021/bi501258u. Epub 2015 Jan 21.
Crystal structures of nickel-dependent superoxide dismutases (NiSODs) reveal the presence of a H-bonding network formed between the NH group 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 intrasubunit 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 density functional theory calculations. The X-ray crystal structure of H1A-NiSOD, which lacks the apical ligand entirely, reveals that in the absence of the Glu17-His1 H-bond, the active site is disordered. Characterization of this variant 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 retains 4% of wild-type (WT) NiSOD activity. Three other mutations were designed to preserve the apical imidazole ligand but perturb the H-bonding network: R47A-NiSOD, which lacks the intramolecular H-bonding interaction; E17R/R47A-NiSOD, which retains the intramolecular H-bond but lacks the intermolecular 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 to probe the nickel site structure, kinetic studies employing pulse-radiolytic production of superoxide, and electron paramagnetic resonance to assess the Ni redox activity. The results indicate that in addition to the roles in redox tuning suggested on the basis of previous computational studies, the Glu17-His1 H-bond plays an important structural role in the proper folding of the "Ni-hook" motif that is a critical feature of the active site.
镍依赖性超氧化物歧化酶（NiSODs）的晶体结构显示，在六聚体酶中，来自His1的顶端咪唑配体的NH基团与相邻亚基的Glu17羧酸盐之间形成了一个氢键网络。这种相互作用得到了Glu17和Arg47之间另一个亚基内氢键的支持。在本研究中，制备了四种突变型NiSOD蛋白，以通过实验评估这个氢键网络的作用，并将结果与密度泛函理论计算的先前预测进行比较。完全缺乏顶端配体的H1A-NiSOD的X射线晶体结构表明，在没有Glu17-His1氢键的情况下，活性位点是无序的。使用X射线吸收光谱（XAS）对该变体进行表征表明，Ni(II) 结合在预期的N2S2平面配位位点。尽管存在这些结构扰动，H1A-NiSOD变体仍保留了野生型（WT）NiSOD活性的4%。设计了另外三个突变以保留顶端咪唑配体但扰动氢键网络：缺乏分子内氢键相互作用的R47A-NiSOD；保留分子内氢键但缺乏分子间Glu17-His1氢键的E17R/R47A-NiSOD；以及缺乏两种氢键相互作用的E17A/R47A-NiSOD。这些变体通过多种技术进行表征，包括用于探测镍位点结构的XAS、采用超氧化物脉冲辐射产生的动力学研究以及用于评估Ni氧化还原活性的电子顺磁共振。结果表明，除了先前计算研究表明的在氧化还原调节中的作用外，Glu17-His1氢键在“Ni钩”基序的正确折叠中起着重要的结构作用，而“Ni钩”基序是活性位点的关键特征。
