SINGLE CRYSTAL XAS STUDIES OF NITROGENASE PROTEINS

固氮酶蛋白的单晶 XAS 研究

• 批准号: 8169950
• 负责人: BRITT HEDMAN
• 金额: $ 0.34万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169950
• 关键词: Address; Anabolism; Biological Process; Catalysis; Computer Retrieval of Information on Scientific Projects Database; Crystallography; Detection; Electronics; Enzymes; Funding; Grant; Incubated; Institution; Metals; Molybdoferredoxin; Nitrogen; Nitrogen Fixation; Nitrogenase; Proteins; Research; Research Personnel; Resources; Science; Site; Source; Structure; System; Techniques; Tertiary Protein Structure; United States National Institutes of Health; electron donor; inhibitor/antagonist; interstitial; molecular vector; protein structure; vector

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The nitrogenase enzyme system catalyzes the essential process of biological nitrogen fixation. It is composed of the MoFe protein, where substrate reduction occurs, and the Fe protein, which is the specific electron donor to the MoFe protein. FeMoco, a [MoFe7S6X] cluster in the MoFe protein, is believed to be the site of nitrogen catalysis. The central low-Z atom, X, at the core of FeMoco was recently discovered by crystallography (O. Einsle, et al. Science 297, 1696 (2002)). The presence of an interstitial atom in FeMoco raises new questions about the structure, biosynthesis, and reactivity of this cluster that we hope to address using single crystal XAS. Polarized single crystal XAS has the advantage of providing both local structural and electronic detail about a metal site in a protein that is related to a known protein tertiary structure and, further, can be used to selectively enhance specific molecular vectors through crystallographic alignment with the beam polarization vector. We have preliminary results that indicate the feasibility of applying this technique to interrogate the central atom of FeMoco. The aim of this study is to further develop and utilize single crystal XAS for the study of important questions in the nitrogenase system including (1) detection and quantification of an interstitial atom in FeMoco; (2) determination of FeMoco structure, especially with regard to the presence of an interstitial atom, in the absence of N2 catalysis through the study of a catalytically inactive MoFe protein; (3) correlation of the Fe-Fe distances in the all-ferrous Fe protein with the global protein structure to gain an understanding of how this distinct cluster may function in catalysis; and (4) elucidation of the mechanism of substrate and inhibitor interaction with FeMoco through the study of radiolytically reduced, substrate-incubated MoFe proteins.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 固氮酶系统催化生物固氮的基本过程。它由发生底物还原的 MoFe 蛋白和 Fe 蛋白组成，Fe 蛋白是 MoFe 蛋白的特异性电子供体。 FeMoco 是 MoFe 蛋白中的一个 [MoFe7S6X] 簇，被认为是氮催化的位点。 FeMoco 核心的中心低 Z 原子 X 最近通过晶体学发现（O. Einsle, et al. Science 297, 1696 (2002)）。 FeMoco 中间隙原子的存在提出了有关该簇的结构、生物合成和反应性的新问题，我们希望使用单晶 XAS 来解决这些问题。偏振单晶 XAS 的优点是可以提供与已知蛋白质三级结构相关的蛋白质中金属位点的局部结构和电子细节，此外，还可以通过与光束偏振矢量的晶体学排列来选择性增强特定分子矢量。我们的初步结果表明应用该技术来探究 FeMoco 中心原子的可行性。本研究的目的是进一步开发和利用单晶XAS来研究固氮酶系统中的重要问题，包括（1）FeMoco中间隙原子的检测和定量； (2) 在没有 N2 催化的情况下，通过研究无催化活性的 MoFe 蛋白来确定 FeMoco 结构，特别是间隙原子的存在； (3) 将全铁 Fe 蛋白中的 Fe-Fe 距离与整体蛋白质结构相关联，以了解这个独特的簇如何在催化中发挥作用； (4) 通过研究辐射分解还原、底物孵育的 MoFe 蛋白，阐明底物和抑制剂与 FeMoco 相互作用的机制。