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

mn -取代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，Fe-sub-Mn-SOD和Mn-sub-(Fe)SOD是由于蛋白质所施加的E（调谐）与适合金属离子的E（调谐）之间的不匹配而无活性。通过共振拉曼光谱和ESEEM对两种不同蛋白质中配位溶剂质子化程度的实验测定，将表明蛋白质通过操纵配位溶剂质子化状态来调节E(E)的程度。突变体（Fe）SOD含有Mn- sods的保守Gln特征，突变体（Mn）SOD含有Fe- sods的Gln特征，在每种金属离子结合的情况下，对突变体（Mn）SOD进行光谱和电化学研究，将提供Mn- sods和Fe- sods之间这种保守氨基酸差异对E（）的影响的互惠定量。通过15N NMR直接或间接的ESEEM观察保守Gln侧链，可以探测到该残基与SOD催化活性的关键成分Tyr 34和配位溶剂之间的氢键极性。（Mn）SOD的随机诱变和在铁而不是锰存在下的活性筛选将提供一个无偏的“转换”SOD的选择，这些SOD具有与铁获得的活性。氨基酸序列测定和生物物理和电化学表征将揭示哪些氨基酸赋予Fe活性，通过何种化学机制以及以何种代价与Mn活性。最后，Mn-SOD和Fe-sub-(Mn)SOD的x射线晶体结构将显示蛋白质结构如何依赖于金属离子身份。因此，这项工作将确定控制蛋白质中金属离子活性的一般原理，以及SOD中特定氨基酸如何实现它们的例子。锰和铁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特异性活性。比较非活性Fe-取代-(Mn)SOD和活性Fe-SOD，以及非活性Mn-亚（Fe）SOD和活性Mn-SOD之间的差异应突出可能与活性相关的差异。预计现有酶中的金属离子取代将为获得具有有用性能的新酶提供一条快速途径。因此，了解决定结合金属离子活性的不同蛋白质因子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. ***