SINGLE CRYSTAL XAS STUDIES OF HEME CATALYSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们计划利用单晶XAS来研究两种具有代表性的含血红素酶-恶臭假单胞菌的细胞色素P450和酿酒酵母的细胞色素c过氧化物酶。恶臭假单胞菌的细胞色素P450催化樟脑的区域和立体特异性羟基化，酿酒酵母的细胞色素c过氧化物酶催化过氧化氢的双电子氧化。像大多数铁-血红素酶一样，这些酶被认为是通过几个铁-氧代中间体，包括氧铁基态(化合物I)，作为其催化循环的一部分。虽然这些酶已经通过结晶学和光谱学进行了广泛的表征，但关于它们的活性中间体的性质仍然存在问题。通过使用Fe K-EDGE单晶XAS检测这些酶的稳定、亚稳态和辐射还原状态，我们计划通过EXAFS分析来表征它们的催化中间体的结构，同时通过分析边缘区域来确定电子态。单晶XAS可以用于各向异性体系中，通过与光束偏振向量的晶体排列来选择性地增强特定的分子向量，从而提供关于分子的增强的几何和电子细节。在细胞色素P450的研究中，极化的XAS将被用来分别探测Fe-底物氧的轴向相互作用和Fe-近端配体的相互作用，而不是Fe-血红素的相互作用。通过1)极化单晶XAS研究细胞色素P450在静息状态下的野生型和效应结合构象，以及潜在地在樟脑孵育、化学氧化/还原、氧化和辐射还原产生的其他六种形式中的构象；以及2)单晶XAS研究铁静止态、铁基化合物I状态和化合物I状态辐射生成的产物中细胞色素C过氧化物酶的单晶XAS研究。

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.