Electronic Structure of Heme Enzyme Intermediates from Resonant Inelastic X-ray Scattering and L-Edge X-ray Absorption Spectroscopy

项目总结/摘要 非血红素酶（NHE）及其活性电子轨道的研究已经做了大量的工作。 氧的活化。与NHE类似，含血红素的酶在生物圈中发挥着多种作用， 对人体健康影响很大的因素：解毒，氧气运输和激素合成。重要的是血红素 酶被理解为与NHE相似的程度，然而，用于表征NHE电子的工具 Fe的共价性，例如磁性圆二色性光谱，不适用于血红素， 高度离域的赤道卟啉环，模糊了铁原子的共价性。的共价性 血红素位置的低价轨道，积极地调节中间体在生物学中的功能。直接 探测血红素酶中的Fe中心，该项目将使用相对较新的共振非弹性X射线散射 （RIXS）光谱。1 s2 p RIXS使用K边缘、硬X射线、入射光子并检测随后的2 p到 1 s空穴填充导致与L边缘X射线吸收光谱（XAS）相同的最终状态-具有不同的 选择规则。RIXS产生关于前沿分子轨道（FMO）的电子结构的信息， 特别是d轨道共价，这对研究血红素的氧活化至关重要，但没有缺点 软X射线L边XAS固有的缺点，如高真空要求、高样品浓度， 荧光抑制然而，一种名为TES的新的超低噪声探测器将最终允许Fe L边缘XAS 稀释的酶样品的光谱，并将1 s2 p RIXS与L边XAS相关联将提供差示轨道 前线分子轨道的共价性是反应性的关键。最初，受训者将应用RIXS和L- 边缘XAS非血红素和血红素模型复合物，特别是众所周知的非血红素模型将允许 将实验观察到的4p轨道混合发展到RIXS光谱的模拟中。受训者 然后将探讨Fe=O键的变化时，从非血红素环境中的酶。 使用L边XAS和RIXS，细胞色素p450化合物II的反应轨道将揭示驱动力 羟基化的“反弹机制”。接下来，本研究将探讨Fe=O键在 转化为化合物I自由基阳离子中间体和对H原子提取的影响。最后， 化合物I的前线分子轨道将在血红素中的不同跨轴连接上进行比较 内切酶轴向配体（组氨酸、半胱氨酸和酪氨酸）的这种互换将定量鉴定， 目前，对血红素电子结构的“推”和“拉”效应的定义不严格， 乳沟本项目将进一步加深血红素和非血红素中铁氧化学的基础知识， 内切酶为Fe=O键的性质和反应性提供了新的见解，卟啉的作用 在血红素中的作用，以及如何在没有这种电子汇的NHE中适应。这项工作还将 开发1 s2 p RIXS和TES检测L-edge XAS在生物无机系统上的方法和建模。

Project Summary/Abstract Much work has been performed to study reactive electron orbitals in nonheme enzymes (NHE) and their activation of oxygen. Similar to NHEs, heme-containing enzymes play a diverse set of roles in the biosphere and factor heavily to human health: detoxification, oxygen transport, and hormone synthesis. It is important that heme enzymes be understood to a similar extent as NHEs, however the tools used to characterize NHE electron covalency of Fe, such as magnetic circular dichroism optical spectroscopy, are not as applicable to hemes due to the highly delocalized equatorial porphyrin ring that obscures the covalency of the Fe atom. The covalency of the low-lying valence orbitals of the heme site, actively tunes intermediates for their function in biology. To directly probe the Fe center in heme enzymes, this project will use the relatively new resonant inelastic X-ray scattering (RIXS) spectroscopy. 1s2p RIXS uses K-edge, hard X-ray, incident photons and detects the subsequent 2p to 1s hole-filling resulting in the same final state as L-edge X-ray absorption spectroscopy (XAS) – with different selection rules. RIXS yields information about the electronic structure of the frontier molecular orbitals (FMOs), specifically d orbital covalency, critical to studying oxygen activation by hemes, however without the weaknesses inherent in soft X-ray L-edge XAS such as high vacuum requirements, high sample concentrations, and fluorescence inhibition. However, a new ultra-low noise detector called the TES will finally allow Fe L-edge XAS spectra of dilute enzyme samples, and correlating 1s2p RIXS with L-edge XAS will afford the differential orbital covalency of the frontier molecular orbitals that are key to reactivity. Initially, the trainee will apply RIXS and L- edge XAS to nonheme and heme model complexes, particularly the well-understood nonheme models will allow development of the experimentally observed 4p orbital mixing into the simulation of RIXS spectra. The trainee will then explore the change of the Fe=O bond when going from a nonheme to a heme environment in an enzyme. With L-edge XAS and RIXS the reactive orbitals of cytochrome p450 compound II will reveal the driving forces for the “rebound mechanism” of hydroxylation. Next, this study will explore the change in the Fe=O bond upon conversion to the compound I radical cation intermediate and the implications for H-atom abstraction. Finally, the frontier molecular orbitals of compound I will be compared across the different trans axial ligations in heme enzymes. This interchange of the axial ligand (histidine, cysteine, and tyrosine) will quantitively identify the, currently, loosely defined “push” and “pull” effects on the heme electronic structure that allow heterolytic O2 cleavage. This project will further the fundamental knowledge of Fe oxygen chemistry in heme, and nonheme, enzymes. Providing new insights into the nature and reactivity of the Fe=O bond, the role that the porphyrin plays in hemes, and how that is adapted in NHEs where no such electron sink is available. This work will also develop the methods and modelling of 1s2p RIXS and TES detected L-edge XAS on bioinorganic systems.