Biosynthetic Models of Heteronuclear Metalloenzymes in Multi-electron Processes

项目总结/摘要 该项目的总体目标是实现结构的整体理解， 参与多电子氧化还原过程的杂合金属酶的功能， 这是更难研究比homeostransferase，并解决重要的 呼吸和全球氮和硫循环领域的科学问题。 具体来说，我们试图研究为什么一氧化氮中的血红素-非血红素Fe中心 还原酶（NOR）在NO的2 e-还原中有效，允许N-N键形成，而 血红素-Cu氧化酶（HCO）中的血红素-Cu中心精通O2的4 e-还原， O-O键断裂，而两者在亚硫酸盐还原酶中的血红素-Cu中心与W. 琥珀酸中心（SiRA）和同化亚硫酸盐还原酶（SiR）中的血红素-Fe 4S 4中心， 在6 e-还原亚硫酸盐时有效，促进S-O键断裂。为了实现这一目标， 这项提议是基于一个科学前提，即开发一种新的生物合成方法， 使用稳定的、易于生产的和良好表征的血红素蛋白作为支架， 使HCO、NOR、SiRA和SiR的结构和功能模型可以克服 在实地取得进展的关键方法障碍。我们将使用生物合成模型 1）了解血红素-Cu中心如何表现出HCO或SiR活性，2）阐明 负责SiR的催化活性和底物结合亲和力的结构特征，3） 阐明Tyr在HCO和SiR活性中的作用; 4）研究血红素的作用 HCO、NOR和SiR活性的辅助因子。为了确保科学的严谨性，我们将使用活性 作为我们的项目设计和光谱，晶体学和计算的指导， 技术来描述我们的模型。 实现上述目标将导致对结构的更深入了解， HCO，NOR，SiRA和SiR的功能可能很难通过研究 单独的天然酶。能够将不同的异质金属中心放置到 相同的蛋白质支架提供了对四种蛋白质之间的相似性和差异的深入了解， 杂合金属酶在这样做的过程中，该项目将增进对以下方面的了解： 金属蛋白质的结构，功能和设计一般，作为指导原则获得 从这些研究将适用于广泛的金属酶的重要性， 人体健康

Project Summary/Abstract The overall goal of the project is to achieve holistic understanding of structure and function of heteronuclear metalloenzymes involved in multi-electron redox processes, which are more difficult to study than homonuclear enzymes, and to address important scientific issues in the fields of respiration and the global nitrogen and sulfur cycles. Specifically, we seek to investigate why a heme-nonheme Fe center in nitric oxide reductase (NOR) is effective at 2e- reduction of NO, allowing N-N bond formation, whereas a heme-Cu center in heme-Cu oxidase (HCO) is proficient at 4e- reduction of O2, enabling O-O bond cleavage, while both a different heme-Cu center in sulfite reductase from W. succinogenes (SiRA) and a heme-Fe4S4 center in assimilatory sulfite reductases (SiR) are efficient at 6e- reduction of sulfite, promoting S-O bond cleavage. To achieve this goal, the proposal is based on a scientific premise that developing a novel biosynthetic approach using stable, easy-to-produce, and well-characterized heme proteins as scaffolds for making structural and functional models of HCO, NOR, SiRA and SiR can overcome critical methodological barriers to progress in the field. We will use the biosynthetic models to 1) Understand how a heme-Cu center can exhibit either HCO or SiR activity, 2) elucidate structural features responsible for catalytic activity and substrate binding affinity of SiR, 3) clarify the roles of Tyr in HCO and SiR activities, and 4) investigate the roles of heme cofactors in HCO, NOR and SiR activities. To ensure scientific rigor, we will use activity as guidance for our project design and spectroscopic, crystallographic and computational techniques to characterize our models. Achieving the above goals will result in deeper understanding of the structure and function of HCO, NOR, SiRA and SiR that may be very difficult to achieve by studying the native enzymes alone. The ability to place different heteronuclear metal centers into the same protein scaffold offers insight into similarities and differences between the four heteronuclear metalloenzymes. In doing so, the project will advance the knowledge of metalloprotein structure, function, and design in general, as the guiding principles obtained from these studies will be applicable to a broad range of metalloenzymes important for human health.