Nickel Superoxide Dismutase

无机化学、生物无机化学和金属有机化学项目的这一奖项支持马萨诸塞州大学的迈克尔·马罗尼教授的研究，目的是了解蛋白质在重组天蓝色链霉菌镍超氧化物歧化酶(NiSOD)中创建活性位点的作用。点突变将被用来解决以下问题：1.镍的第二配位球上的残基在优化活性部位的氧化还原电位中起关键作用吗？2.催化所需的四级结构吗？3.活性部位附近保守的酪氨酸残基起什么作用？4.蛋白质结构如何调节活性部位与过氧化氢的相互作用？过氧化氢是一种反应产物，活性部位的还原剂，并容易氧化硫酸盐，如活性部位中发现的半胱氨酸配体？突变的酶将使用一种策略来表征，该策略采用了各种互补的生物物理技术，包括EPR和X射线吸收光谱。突变对催化的影响将通过测量催化速率常数，使用超氧化物的脉冲辐射产生和UV-Vis光谱来研究反应机理，并通过氧化还原滴定评估酶的氧化还原潜力来解决。将使用结晶学进一步研究导致机制受扰的突变。在现有的一些突变的NiSOD中检测到的催化中间体将被冷冻捕获并进行光谱分析。对NiSOD的研究将有助于理解生物镍中心对氧化还原的催化作用。该研究将为各级学生(本科生到博士后)提供化学和生物相结合的多学科培训。

This award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports research by Professor Michael Maroney at the University of Massachusetts to understand the role of the protein in creating the active site in recombinant Streptomyces coelicolor nickel superoxide dismutase (NiSOD). Point mutations will be employed to address the following questions: 1. Do residues in the second coordination sphere of Ni play a critical role in optimizing the redox potential of the active site? 2. Is the quaternary structure required for catalysis? 3. What are the roles of conserved tyrosine residues near the active site? 4. How does the protein structure moderate the interaction of the active site with hydrogen peroxide, a molecule that is a reaction product, an active site reductant, and readily oxidizes thiolates, such as the cysteine ligands found in the active site? Mutant enzymes will be characterized using a strategy that employs a variety of complementary biophysical techniques including EPR and x-ray absorption spectroscopy. The effect of mutations on catalysis will be addressed by measuring the catalytic rate constant and examining the reaction mechanism using pulse radiolytic generation of superoxide and UV-Vis spectroscopy, and by assessing the redox potential of the enzyme using redox titrations. Mutations that result in a perturbed mechanism will be further examined using crystallography. Catalytic intermediates that have been detected in a number of existing mutant NiSODs will be freeze-trapped and examined spectroscopically The study of NiSOD will contribute to the understanding of redox catalysis by biological nickel sites. The research will provide multidisciplinary training at the interface of chemistry and biology for students at all levels (undergraduate through post-doctoral).