Mechanistic and spectroscopic investigation of sulfur-oxidizing non-heme iron enzymes

来自德克萨斯大学阿灵顿分校的布拉德·皮尔斯博士将在该奖项中研究哺乳动物硫醇双加氧酶(TDO)的机制和调控。暂态非血红素铁[Fe-O]中间体的表征历来在生物无机化学领域引起了极大的兴趣。这种强烈的研究努力重点在很大程度上可以解释为大量功能(和结构上)不同的非血红素铁酶，以及它们引发的化学氧化反应所显示的令人难以置信的多功能性。与这类酶的几乎所有成员所表现出的典型的2-His-1-Cxylate面部三联体基序不同，哺乳动物TDO酶的活性部位利用了中性的All-His(3-His)面部三联体基序。此外，TDO酶还有一对不寻常的共价交联半胱氨酸-酪氨酸对，位于非血红素铁活性部位附近。这项提议的一个目标是鉴定和光谱表征在TDO催化的硫醇氧化过程中产生的瞬时中间体。另一个焦点领域是TDO酶活性部位中罕见的转录后半胱氨酸-酪氨酸共价修饰的作用。具体地说，将测定TDO酶的稳态O2偶联效率，以与C93和Y157突变株进行比较。这些结果将与光谱学观察到的活性中心内的特定分子相互作用相关联，以评估外球相互作用在天然TDO催化中的作用。与加氧酶/氧化酶不同，人工合成的非血红素铁模型络合物不能通过底物结合来抑制O2-反应。因此，模型体系对氧化转化的催化作用很差。目前，非血红素铁催化的这一鲜为人知的方面是开发工业上有用的氧气依赖型铁催化剂的最大障碍之一。因此，另一个研究领域将集中在TDO酶通过蛋白质-底物相互作用调节O2-反应的机制。皮尔斯博士的研究具有潜在的技术潜力，因为所获得的结果可以应用于生物启发的氧化转化催化剂的设计。该奖项将对德克萨斯大学阿灵顿分校成为一所全国性研究型大学和培养准备充分的STEM学生的战略计划做出重大贡献。此外，鉴于该大学的第一代学生人数众多，通过资助本科生和研究生的研究机会，可以对增加STEM领域的多样性产生重大影响。指导未来的科学家是一个重要的优先事项，这个奖项将为当地的高中生和大学的本科生提供持续的研究机会和支持。拟议研究的跨学科性质将为各个层次的学生(高中、本科生和研究生)提供广泛的研究培训。

In this award from the Chemistry of Life Processes Program in the Division of Chemistry, Dr. Brad Pierce, from the University of Texas at Arlington, will study the mechanism and regulation of mammalian thiol dioxygenase (TDO) enzymes. Characterization of transient non-heme iron [Fe-O] intermediates has historically attracted considerable interest within the area of bioinorganic chemistry. This intense focus of research efforts can largely be explained by the vast number of functionally (and structurally) diverse non-heme iron enzymes and the incredible versatility exhibited in chemical oxidations they initiate. In contrast to the canonical 2-His-1-carboxylate facial triad motif exhibited by nearly all members of this enzyme class, the active site of mammalian TDO enzymes utilize a neutral, all-His (3-His) facial triad motif. Furthermore, TDO enzymes also have an unusual covalently cross-linked cysteine-tyrosine pair in close proximity to the non-heme iron active site. One objective for this proposal is to identify and spectroscopically characterize transient intermediates produced during TDO-catalyzed thiol-oxidation. An additional area of focus is the role of a rare post-transcriptional cysteine-tyrosine covalent modification within the active site of TDO enzymes. Specifically, steady-state O2-coupling efficiency of TDO enzymes will be determined for comparison to C93 and Y157 mutants. These results will be correlated to specific molecular interactions within the active site as observed by spectroscopy to evaluate the role of outer-sphere interactions on native TDO catalysis. In contrast to oxygenase/oxidase enzymes, synthetic non-heme iron model complexes are unable to gate O2-reactivity through substrate-binding. As a consequence, model systems are poor catalysts for oxidative transformations. Currently, this poorly understood aspect of non-heme iron catalysis represents one of the most significant hurdles in the development of industrially useful O2-dependent iron catalysts. Therefore, an additional area of investigation will focus on the mechanism by which TDO enzymes modulate O2-reactivity through protein-substrate interactions. Dr. Pierce's research is potentially technology-enabling in that results obtained could be applied to the design of biologically inspired catalysts for oxidative transformations. The award will contribute significantly to the University of Texas at Arlington's strategic plan of becoming a national research university and a source of well-prepared STEM students. Furthermore, given the high population of first-generation students at the University, a significant impact can be made toward increasing diversity within STEM fields by sponsoring undergraduate and graduate research opportunities. Mentoring future scientists is a significant priority, and this award will provide continued research opportunities and support for both local area high school students and undergraduates at the University. The interdisciplinary nature of the proposed research will provide a wide breadth of research training for students at every level (high school, undergraduate, and graduate).