SINGLE CRYSTAL XAS STUDIES OF HEME CATALYSIS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 我们计划利用单晶XAS研究两个代表性的血红素含酶？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，和来自酿酒酵母的细胞色素c过氧化物酶，其催化过氧化氢的双电子氧化。与大多数铁血红素酶一样，这些酶被认为通过几种铁氧中间体，包括氧铁基状态（化合物I），作为其催化循环的一部分。虽然，这些酶已被广泛的晶体学和光谱学的特点，问题仍然存在关于其活性中间体的性质。通过检查稳定的，亚稳态的，这些酶的放射性还原状态，使用铁K-边缘单晶XAS，我们计划通过EXAFS分析伴随着电子状态的测定，通过分析的边缘区域来表征其催化中间体的结构。单晶XAS可用于各向异性系统中，以通过与光束偏振矢量的晶体学对准来选择性地增强特定的分子矢量，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研究中，极化XAS将用于分别探测轴向Fe-底物氧和Fe-近端配体相互作用与Fe-血红素相互作用。本文概述的程序应该通过1）细胞色素P450在静止状态下和潜在地在由樟脑孵育、化学氧化/还原、氧合和辐射分解还原产生的六种其它形式下的野生型和效应物结合构象的极化单晶XAS研究提供对血红素酶催化的详细理解;和2）在铁静止态、铁基化合物I态和化合物I态的辐射分解产物中的细胞色素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.