Conformational Coupling and the Basis for Metal Ion Specificity in Superoxide Dismutase

本研究将以含铁锰超氧化物歧化酶（SOD）为模型体系，利用核磁共振波谱（NMR）的独特能力，直接观察溶液中的蛋白质基团和监测结构。目的是阐明金属离子位点与SOD蛋白在底物与金属离子类似结合时的构象相互作用，并确定为什么FeSOD蛋白与Mn结合而不是Fe结合无活性，MnSOD蛋白与Fe结合而不是Mn结合无活性。同位素编辑核磁共振将与氨基酸特异性同位素标记一起使用，以产生仅显示标记氨基酸信号的急剧简化的光谱。比较有或没有底物类似叠氮化物结合的SOD的光谱将识别受叠氮化物结合影响的氨基酸，并指出蛋白质通过何种机制对金属离子的事件作出构象响应。通过监测结构域或亚基之间界面处的氨基酸，可以确定构象变化是否涉及构象变化的潜在一般机制，包括：(1)金属离子桥接的结构域的相对运动，(2)亚基的相对运动和(3)通过氢键网络的构象效应的传播。这些研究建立在SOD已知结构的基础上，利用核磁共振直接观察质子的独特能力，提供它们参与氢键的信息，并提供不受晶体包装约束的溶液中蛋白质结构和动力学的定性和定量信息。这项工作还将确定为什么含有错误金属离子的sod是无活性的，通过评估它们对单个元素的催化能力，包括每个半反应，还原中点电位，质子捐赠，底物类似物结合和活性位点结构，从而明确催化无能的化学原因。位点定向和随机诱变将用于将MnSOD蛋白转化为FeSOD。这些sod的表征将揭示哪些氨基酸变化是重要的，这些氨基酸变化在多大程度上支持与错误金属的活性，以及与正确金属的活性的代价是什么。金属酶将金属中心的反应活性与酶的精细特异性和控制相结合。含铁超氧化物歧化酶（FeSODs）和相应的含锰超氧化物歧化酶（MnSODs）是催化超氧化物转化为双氧和过氧化氢的金属酶。SOD相对较小，非常稳定和可溶，但也体现了金属酶催化的两个核心特征：金属位点和蛋白质被认为会影响彼此的结构，每种类型的SOD虽然可以结合铁或锰，但只对其中一种有活性。因此，酶的结构和活性反映了蛋白质和金属离子之间的相互作用，这些相互作用是金属离子特异性的。这项研究的目标是：(1)确定小分子与金属离子结合对蛋白质的影响程度，(2)根据组成蛋白质结构的模块的相对运动来描述蛋白质结构的变化。这项工作还将确定为什么MnSOD蛋白对铁结合不活跃，而FeSOD蛋白对锰结合不活跃。这种对金属酶催化活性所必需的蛋白质-金属合作的新认识将推动正在进行的设计或修饰金属酶以催化药物合成，废物解毒和化学传感器中的有用反应的努力。* * *

9418181 Miller The research will employ Fe and Mn-containing superoxide dismutase (SOD) as a model system and exploit the unique ability of nuclear magnetic resonance spectroscopy (NMR) to directly observe protein groups and monitor structure in solution. The objectives are to elucidate the conformational interplay between the metal ion site and the protein of SOD upon substrate analog binding to the metal ion, and to determine why FeSOD protein is inactive with Mn bound instead of Fe, and MnSOD protein is inactive with Fe bound instead of Mn. Isotope edited NMR will be used in conjunction with amino acid-specific isotopic labeling to produce drastically simplified spectra showing signals of labeled amino acids only. Comparisons of spectra of SOD with and without the substrate analog azide bound will identify amino acids affected by azide binding and indicate by what mechanisms the protein is conformationally responsive to events at the metal ion. By monitoring amino acids at the interfaces between domains or subunits, it will be possible to determine whether the conformation change involves potentially general mechanisms for conformational change including (1) relative motions of domains bridged by the metal ion, (2) relative motions of subunits and (3) propagation of conformational effects via hydrogen bonding networks. These studies build on the known structure for SOD and exploit the unique ability of NMR to directly observe protons, provide information on their participation in hydrogen bonds and provide qualitative and quantitative information on both structure and dynamics of proteins in solution unconstrained by crystal packing. The work will also determine why SODs containing the wrong metal ion are inactive, by evaluating their competence for individual elements of catalysis including each of the half reactions, the reduction midpoint potential, proton donation, substrate analog binding and active site structure, thus specifying the chemical reaso n for catalytic incompetence. Site-directed and random mutagenesis will be used to convert MnSOD protein into a FeSOD. Characterization of these SODs will reveal what amino acid changes are important, the extent to which these amino acid changes support activity with the wrong metal, and at what cost to activity with the correct metal. %%% Metalloenzymes combine the reactivity of metal centers with the exquisite specificity and control of enzymes. The Fe-containing superoxide dismutases (FeSODs) and the homologous Mn-containing superoxide dismutases (MnSODs) are metalloenzymes that catalyze the conversion of superoxide to dioxygen and hydrogen peroxide. SOD is relatively small, very stable and soluble but also embodies two central features of metalloenzyme catalysis: the metal site and the protein are believed to affect each other's structure, and each type of SOD, while able to bind either Fe or Mn, is only active with one. Thus, the structure and activity of the enzyme reflect interactions between the protein and the metal ion, and these interactions are metal ion specific. The goal of the research is (1) to determine how much of the protein is affected by binding of small molecules to the metal ion, and (2) to describe the protein structural change in terms of relative movement of the modules that make up the protein structure. The work will also determine why MnSOD protein is not active with Fe bound and FeSOD protein is not active with Mn bound. This new understanding of the protein-metal cooperation that is necessary for metalloenzyme catalytic activity will advance ongoing efforts to design or modify metalloenzymes to catalyze useful reactions in drug synthesis, waste detoxification and chemical sensors. ***