Second-Sphere Influences on Oxygen Activation by Non-Canonical Heme Oxygenases

项目摘要 这项研究计划阐明了两种非规范的血红素加氧酶是如何调节电子的 结构，以催化新的血红素-氧化学，并开发新的研究工具，以 实现这一目标。MhuD和IsdG活性中心内的第二球相互作用调谐电子 实现区域特异性卟啉氧化的过氧铁中间体的结构和反应活性 没有在典型的血红素加氧酶中发现的保守水团的帮助。观测到的反应性 不能归因于从酶的活性部位和血红素的电子结构的纯空间控制 对于纯理论方法来说太复杂了，所以研究团队紧密地结合了光谱学 用计算机模拟表征MhuD和MhuD的新的血红素-氧反应性 IsdG.为了达到本研究项目的目的，课题组开发了新的光谱技术。 实验，包括确定亚铁血红素电子构型的新方法和 血红素结合的氧气的取向。研究小组还开发了新的计算模型来帮助 光谱分析数据，包括卟啉复杂影响的一般理论模型 对血红素电子吸收光谱的影响。这项研究计划的结果对 通过阐明一种新的反应途径和开发新的研究工具， 和公共卫生，通过获得为开发新抗生素奠定基础的知识。 这个研究项目的目标是通过刻画四秒钟的影响来实现的- MhuD和IsdG活性中心中的球相互作用对其结构、电子结构和反应活性的影响 非典型血红素加氧酶催化的血红素降解的两个关键中间体。的第一个目标是 研究小组将确定MhuD和IsdG的变体，这些变体的功能因活性部位的变化而改变。这 目的是通过使用光谱分析来鉴定具有改变功能的变体，以及额外的 对这些变体进行光谱表征，以确定这些取代是否改变了 多肽二级结构。研究小组的第二个目标是确定这些第二个球体如何 具有改变的功能和不改变的二级结构的变体扰乱铁(氢)过氧体中间体。 这一目的是通过使用光学光谱学来快速识别具有扰动底物的变体来实现的 电子结构，并利用磁谱学和理论计算详细描述了 电子结构变化及其对血红素-氧气反应性的影响。最后，本研究的第三个目的 该团队的目的是阐明MhuD和IsdG活性位点如何调节中羟基血红素的反应性。这个 研究小组使用空气敏感设备来准备这些活性中间体，用于光谱和 机械化的特征。最终，这项研究计划使用了生化、光谱和 用于表征非典型血红素加氧酶催化的两个氧化反应的计算工具。

Project Summary This research program elucidates how two non-canonical heme oxygenases tune the electronic structure of heme in order to catalyze novel heme–dioxygen chemistry, and develops new research tools to achieve this objective. Second-sphere interactions within the MhuD and IsdG active sites tune the electronic structure and reactivity of a ferric–(hydro)peroxo intermediate to achieve regiospecific porphyrin oxygenation without the aid of the conserved water cluster found in canonical heme oxygenases. The observed reactivity cannot be attributed to purely steric control from the enzyme active sites, and the electronic structure of heme is far too complex for a purely theoretical approach, so the research team closely integrates spectroscopic characterization with computational modelling to understand the novel heme–dioxygen reactivity of MhuD and IsdG. In order to achieve the aims of this research project, the research team develops new spectroscopic experiments, including new approaches to determine the electron configuration of ferric heme and the orientation of heme-bound dioxygen. The research team also develops new computational models to aid analysis of the spectroscopic data, including a general theoretical model for the complex influence of porphyrin ruffling on the electronic absorption spectrum of heme. The results from this research program benefit both the fundamental heme community, by elucidating a novel reactive pathway and developing new research tools, and public health, by acquiring knowledge that lays the foundation for the development of new antibiotics. The objectives of this research program are achieved by characterizing the influence of four second- sphere interactions in the MhuD and IsdG active sites on the structure, electronic structure, and reactivity of two critical intermediates of non-canonical heme oxygenase-catalyzed heme degradation. The first aim of the research team is to identify variants of MhuD and IsdG with altered function due to active site changes. This aim is achieved by employing spectroscopic assays to identify variants with altered function, and additional spectroscopic characterization of these variants to determine whether these substitutions change the polypeptide secondary structure. The research team’s second aim is to determine how these second sphere variants with altered function and unaltered secondary structure perturb the ferric–(hydro)peroxo intermediate. This aim is achieved by using optical spectroscopy to rapidly identify variants with perturbed substrate electronic structures, and employing magnetic spectroscopies and theoretical calculations to detail the electronic structure changes and their effect on heme–dioxygen reactivity. Finally, the third aim of the research team is to elucidate how the MhuD and IsdG active sites tune the reactivity of mesohydroxyheme. The research team employs air-sensitive equipment to prepare these reactive intermediates for spectroscopic and mechanistic characterization. Ultimately, this research program uses biochemical, spectroscopic, and computational tools to characterize two oxygenation reactions catalyzed by non-canonical heme oxygenases.