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

项目摘要/摘要 人们已经做了大量工作来研究非血红素酶(NHE)中的反应电子轨道及其 氧气的激活。与NHEs类似，含亚铁血红素的酶在生物圈和 对人类健康有很大影响的因素：解毒、氧气运输和激素合成。重要的是，血红素 对酶的理解程度与NHEs类似，但用于表征NHEs电子的工具 铁的共价性，如磁性圆二色光谱，由于 到高度离域的赤道卟啉环，它遮盖了铁原子的共价性。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上的功能。直接 探测血红素酶中的铁中心，本项目将使用相对较新的共振非弹性X射线散射 (RIXS)光谱学。1s2p RIXS使用K边硬X射线入射光子，并检测随后的2p到 1S空穴填充导致与L相同的最终状态-边缘X射线吸收光谱(XAS)-不同 选择规则。RIXS提供了关于前线分子轨道(FMO)的电子结构的信息， 特别是d轨道共价性，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边缘X射线吸收光谱的固有特性，如高真空要求、高样品浓度和 荧光抑制。然而，一种名为TES的新的超低噪声探测器最终将允许铁L边缘XAS 稀酶样品的光谱，并将1s2p RIXS与L边XAS关联将提供差示轨道 前线分子轨道的共价性，这是反应性的关键。最初，学员将应用RIXS和L- Edge XAS到非亚铁血红素和亚铁血红素模型复合体，特别是众所周知的非亚铁血红素模型将允许 将实验观测到的4p轨道混合发展到RIXS谱的模拟中。实习生 然后将探索在酶中从非血红素环境到血红素环境时Fe=O键的变化。 利用L-EDGE XAS和RIXS，细胞色素P450化合物II的反应轨道将揭示驱动力 关于羟化的“反弹机制”。下一步，这项研究将探索Fe=O键在 向化合物I自由基阳离子中间体的转化及其对H原子抽提的启示。最后， 化合物I的前线分子轨道将通过不同的血红素跨轴连接进行比较 酵素。轴向配体(组氨酸、半胱氨酸和酪氨酸)的这种交换将定量地识别， 目前，对血红素电子结构的松散定义的“推动”和“拉动”效应允许异源分解O2 乳沟。该项目将进一步加深对血红素和非血红素中铁氧化学的基础知识， 酵素。提供了对Fe=O键的性质和反应活性的新见解，即卟啉 在血红素中发挥作用，以及如何在没有这样的电子接收器的NHEs中改编。这项工作还将 建立了生物无机体系中1s2p RIXS和TES检测L边缘XAS的方法和模型。

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.