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

项目概要 该研究项目阐明了两种非经典血红素加氧酶如何调节电子 血红素的结构，以催化新型血红素-双氧化学，并开发新的研究工具 实现这一目标。 MhuD 和 IsdG 活性位点内的第二球体相互作用调节电子 铁-（氢）过氧中间体的结构和反应性以实现区域特异性卟啉氧化 没有典型血红素加氧酶中发现的保守水簇的帮助。观察到的反应性 不能归因于酶活性位点的纯粹空间控制以及血红素的电子结构 对于纯理论方法来说太复杂了，因此研究团队将光谱学紧密结合起来 通过计算模型进行表征，以了解 MhuD 的新型血红素-双氧反应性和 IsdG。为了实现该研究项目的目标，研究团队开发了新的光谱 实验，包括确定铁血红素电子构型的新方法和 血红素结合双氧的方向。研究团队还开发了新的计算模型来帮助 光谱数据分析，包括卟啉复杂影响的一般理论模型 血红素电子吸收光谱的波动。该研究计划的结果使双方受益 基本血红素社区，通过阐明新的反应途径并开发新的研究工具， 和公共卫生，通过获取为开发新抗生素奠定基础的知识。 该研究计划的目标是通过表征四个第二- MhuD 和 IsdG 活性位点的球体相互作用对结构、电子结构和反应性的影响 非经典血红素加氧酶催化血红素降解的两个关键中间体。该项目的第一个目标是 研究小组的目标是识别由于活性位点变化而导致功能改变的 MhuD 和 IsdG 变体。这个 通过采用光谱分析来识别具有改变功能的变体来实现这一目标，以及额外的 这些变体的光谱表征以确定这些替代是否改变了 多肽二级结构。研究小组的第二个目标是确定第二个领域如何 功能改变和二级结构未改变的变体会扰乱铁-（氢）过氧中间体。 这一目标是通过使用光谱法快速识别基底受到扰动的变体来实现的 电子结构，并采用磁谱和理论计算来详细描述 电子结构变化及其对血红素-氧反应性的影响。最后，本研究的第三个目标 该团队的目标是阐明 MhuD 和 IsdG 活性位点如何调节内消旋羟基血红素的反应性。的 研究小组采用空气敏感设备来制备这些反应中间体，用于光谱和 机械表征。最终，该研究计划使用生物化学、光谱和 用于表征非典型血红素加氧酶催化的两种氧合反应的计算工具。

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.