SINGLE CRYSTAL XAS STUDIES OF HEME CATALYSIS

血红素催化的单晶 XAS 研究

• 批准号: 8169949
• 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/8169949
• 关键词: Binding; Camphor; Catalysis; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Crystallography; Cytochrome P450; Cytochrome c Peroxidase; Electronics; Electrons; Enzymes; Funding; Grant; Heme; Hydrogen Peroxide; Hydroxylation; Institution; Ligands; Molecular Conformation; Nature; Oxidation-Reduction; Oxygen; Pseudomonas putida; Research; Research Personnel; Resources; Rest; Saccharomyces cerevisiae; Source; Spectrum Analysis; Structure; System; United States National Institutes of Health; molecular vector; oxidation; programs; 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. We plan to utilize single crystal XAS to study two representative heme-containing enzymes?cytochrome P450 from Pseudomonas putida, which catalyzes the regio- and stereo-specific hydroxylation of camphor, and cytochrome c peroxidase from Saccharomyces cerevisiae, which catalyzes the two-electron oxidation of hydrogen peroxide. Like most Fe-heme enzymes, these are thought to go through several Fe-oxo intermediates, including an oxy-ferryl state (compound I), as part of their catalytic cycle. Although, these enzymes have been extensively characterized by both crystallography and spectroscopy, questions still remain about the nature of their reactive intermediates. By examining stable, metastable, and radiolytically reduced states of these enzymes using Fe K-edge single crystal XAS, we plan to characterize the structure of their catalytic intermediates through EXAFS analysis concomitant with electronic state determination through analysis of the edge region. Single crystal XAS can be used in anisotropic systems to selectively enhance specific molecular vectors through crystallographic alignment with the beam polarization vector, thereby providing enhanced geometric and electronic details about a molecule. In the cytochrome P450 study, polarized XAS will be used to probe the axial Fe-substrate oxygen and Fe-proximal ligand interactions separately from the Fe-heme interactions. The program outlined herein should provide for a detailed understanding of heme enzyme catalysis through 1) the polarized single crystal XAS study of wild-type and effector bound conformations of cytochrome P450 in the resting state and, potentially, in six other forms produced by camphor incubation, chemical oxidation/reduction, oxygenation, and radiolytic reduction; and 2) the single crystal XAS study of cytochrome c peroxidase in the ferric resting-state, ferryl compound I state, and in the radiolytically generated products of the compound I state.

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。子项目及 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。 我们计划利用单晶XAS来研究两种代表性的含血红素酶：来自恶臭假单胞菌的细胞色素P450，其催化樟脑的区域和立体特异性羟基化，以及来自酿酒酵母的细胞色素c过氧化物酶，其催化双电子氧化过氧化氢。与大多数铁血红素酶一样，这些酶被认为会经历几种铁氧中间体，包括氧铁基状态（化合物 I），作为其催化循环的一部分。尽管这些酶已通过晶体学和光谱学进行了广泛的表征，但其反应中间体的性质仍然存在疑问。通过使用 Fe K 边缘单晶 XAS 检查这些酶的稳定、亚稳定和辐射分解还原态，我们计划通过 EXAFS 分析表征其催化中间体的结构，同时通过边缘区域分析确定电子态。单晶 XAS 可用于各向异性系统，通过与光束偏振矢量的晶体排列来选择性增强特定分子矢量，从而提供有关分子的增强的几何和电子细节。在细胞色素 P450 研究中，偏振 XAS 将用于探测轴向 Fe-底物氧和 Fe-近端配体相互作用，与 Fe-血红素相互作用分开。本文概述的程序应通过 1) 对静息状态下的细胞色素 P450 的野生型和效应器结合构象以及可能的樟脑孵育产生的六种其他形式进行偏振单晶 XAS 研究，从而详细了解血红素酶催化作用、化学氧化/还原、氧化和辐射分解还原； 2)三价铁静息态、FeF1化合物I状态以及化合物I状态的放射分解产物中的细胞色素c过氧化物酶的单晶XAS研究。