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

项目摘要 该研究计划阐明了两种非经典的血红素加氧酶如何调节电子 血红素的结构，以催化新的血红素-二氧化学，并开发新的研究工具， 实现这一目标。MhuD和IsdG活性位点内的第二层相互作用调节了电子 铁-（氢）过氧中间体结构和实现区域特异性卟啉氧化的反应性 而不需要在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 不能归因于来自酶活性位点的纯粹空间控制，并且血红素的电子结构 对于纯理论的方法来说太复杂了，所以研究小组紧密结合了光谱学。 表征与计算建模，以了解新的血红素双氧反应性的MhuD和 IsdG。为了实现本研究项目的目标，研究小组开发了新的光谱分析方法， 实验，包括新的方法来确定铁血红素的电子构型和 血红素结合双氧的取向。研究小组还开发了新的计算模型， 光谱数据的分析，包括卟啉的复杂影响的一般理论模型 血红素的电子吸收光谱的起伏。这项研究计划的结果使双方受益。 基本血红素社区，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，通过获得知识，奠定了新的抗生素开发的基础。 这项研究计划的目标是通过表征四个第二的影响， 在MhuD和IsdG活性位点的结构，电子结构和反应性的球体相互作用 非典型血红素加氧酶催化血红素降解的两个关键中间体。第一个目标 研究小组的一个目标是鉴定由于活性位点变化而改变功能的MhuD和IsdG变体。这 目的是通过采用光谱测定来鉴定具有改变的功能的变体，以及另外的 这些变体的光谱表征，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 具有改变的功能和未改变的二级结构的变体扰乱铁-（氢）过氧中间体。 这一目标是通过使用光谱学来快速识别具有扰动基底的变体来实现的 电子结构，并采用磁光谱和理论计算来详细说明 电子结构变化及其对血红素-二氧反应性的影响。最后，本研究的第三个目的 研究小组的任务是阐明MhuD和IsdG活性位点如何调节mesohydroxyheme的反应性。的 研究团队采用空气敏感设备制备这些活性中间体， 机械表征最终，这项研究计划使用生物化学，光谱， 计算工具来表征由非典型血红素加氧酶催化的两个加氧反应。

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.