Understanding the Basis for Metal Ion Specificity in Fe- andMn-Superoxide Dismutases: Learning to Make Bound Metal IonsCatalytically Active

米勒Mn-取代的Fe-超氧化物歧化酶（Mn-sub-（Fe）（SOD））与Mn-SOD的比较将提供一种测试，即由于蛋白质施加的E（tuning）与适合于金属离子的E（tuning）之间的失配，Fe-sub-Mn-SOD和Mn-sub-（Fe）SOD是无活性的。 通过共振拉曼光谱和ESEEM实验测定两种不同蛋白质中配位溶剂的质子化程度将指示蛋白质可以调节E（通过操纵配位溶剂的质子化状态）的程度。 对含有Mn-SOD的保守Gln特征的突变体（Fe）SOD和含有Fe-SOD的Gln特征的突变体（Mn）SOD的光谱和电化学研究，结合每种金属离子，将提供Mn-和Fe-SOD之间的这种保守氨基酸差异对E（）的影响的倒数定量。 通过15 N NMR直接或通过ESEEM间接观察保守的Gln侧链将探测该残基与SOD催化活性的关键成分Tyr 34和配位溶剂的氢键的极性。 随机诱变（Mn）SOD和筛选活性的存在下，铁，但不锰将提供一个公正的选择“转化”SOD已获得活性与铁。 氨基酸序列测定和生物物理和电化学表征将揭示什么氨基酸赋予Fe活性，通过什么化学机制以及以什么代价赋予Mn活性。 最后，Mn-SOD和Fe-sub-（Mn）SOD的X射线晶体结构的解决方案将显示蛋白质结构如何取决于金属离子身份。 因此，这项工作将确定管理蛋白质中金属离子活性的一般原则，以及它们如何通过SOD中的特定氨基酸实现的例子。 Mn和Fe特异性超氧化物歧化酶（SOD）将用于研究蛋白质如何赋予结合金属离子的特异性活性。 比较无活性的Fe-取代的-（Mn）SOD和活性的Fe-SOD，以及无活性的Mn-亚-（Fe）SOD和活性的Mn-SOD，应突出可能与活性相关的差异。 可以预见，金属离子取代现有的酶可以提供一个快速的路线，以新的酶与有用的特性。因此，了解决定结合金属离子活性的不同蛋白质因素对于设计具有所需催化活性的金属酶的能力至关重要。 ***

9728793 Miller Comparison of Mn-substituted Fe-superoxide dismutase (Mn-sub-(Fe)(SOD) with Mn-SOD will provide a test that Fe-sub-Mn-SOD and Mn-sub-(Fe)SOD are inactive due to a mismatch between the E( tuning applied by the protein and that appropriate to the metal ion. Experimental determination of the degree of protonation of the coordinated solvent in the two different proteins by resonance Raman spectroscopy and ESEEM will indicate the extent to which the proteins could modulate E( by manipulating the protonation state of coordinated solvent. Spectroscopic and electrochemical studies of mutant (Fe)SOD containing the conserved Gln characteristic of Mn-SODs and mutant (Mn)SOD containing the Gln characteristic of Fe-SODs, with each of the metal ions bound, will provide reciprocal quantitations of the effects on the E( of this conserved amino acid difference between Mn- and Fe-SODs. Observation of the conserved Gln's side chain directly by 15N NMR or indirectly by ESEEM will probe the polarity of this residue's hydrogen bonds with key ingredients of SOD's catalytic activity: Tyr 34 and the coordinated solvent. Random mutagenesis of (Mn)SOD and screening for activity in the presence of Fe but not Mn will afford an unbiased selection of "convertant" SODs that have acquired activity with Fe. Amino acid sequence determination and biophysical and electrochemical characterizations will then reveal what amino acids confer activity on Fe, by what chemical mechanisms and at what cost to activity with Mn. Finally, solution of the X-ray crystal structures of Mn-SOD and Fe-sub-(Mn)SOD will show how the protein structure depends on the metal ion identity. Thus, the work will identify general principles governing metal ion activity in proteins and examples of how they are implemented by particular amino acids in SOD. Mn- and Fe-specific superoxide dismutases (SODs) will be used to study how proteins confer specific activity on bound metal ions. Comparisons between inactive Fe-substituted-(Mn)SOD an d active Fe-SOD, and between the inactive Mn-sub-(Fe)SOD and active Mn-SOD should highlight differences that may be correlated with activity. It is anticipated that metal ion substitution in existing enzymes may afford a rapid route to novel enzymes with useful properties. Therefore, knowledge of the different protein factors that determine the activity of bound metal ions is crucial to ability to design metallo-enzymes with desired catalytic activity. ***