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

第二领域对非典型血红素加氧酶的氧活化的影响

基本信息

批准号：
9981995
负责人：
Matthew D Liptak
金额：
$ 17.47万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9981995
关键词：
Active Sites Air Antibiotics Azides Biochemical Biochemical Reaction Biological Assay Carbon Chemistry Circular Dichroism Communities Complex Computer Simulation Development Dioxygen Distal Electron Spin Resonance Spectroscopy Electrons Enzymes Equipment Foundations Funding Genus staphylococcus Goals Heme Heme Iron Hemoglobin Human Hydroxylation Investigation Iron Isomerism Knowledge Knowledge acquisition Laboratories Magnetism Measures Methods Modeling Molecular Mycobacterium tuberculosis NMR Spectroscopy Nutrient Optics Oxygen Oxygenases Pathway interactions Peroxidases Pharmaceutical Preparations Porphyrins Procedures Proteins Public Health Reaction Reactive Oxygen Species Reporting Research Research Project Grants Research Project Summaries Spectrum Analysis Staphylococcus aureus Structural Protein Structure Temperature Theoretical model Variant Water absorption analog base computer studies computerized tools density electronic structure experimental study geometric structure heme a human disease insight magnetic field member mycobacterial novel novel strategies pathogen polypeptide programs protein structure spectroscopic data theories tool

项目摘要

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.

项目摘要该研究计划阐明了两个非典型的血红素氧化酶如何调节电子血红素的结构是为了催化新颖的血红素 - 二氧化学，并开发了新的研究工具实现这一目标。 MHUD和ISDG活动位点中的第二球相互作用调整电子铁 - （氢）过氧中间体的结构和反应性，以实现调节特异性卟啉氧合没有在规范血红素氧化酶中发现的保守水簇的帮助。观察到的反应性不能归因于从酶的活性位点纯粹的空间控制，以及血红素的电子结构对于纯粹的理论方法来说太复杂了，因此研究团队紧密整合了光谱镜通过计算建模进行表征，以了解Mhud和Mhud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镜实验，包括确定铁血红素和电子构型的新方法血红素结合的二恶英的方向。研究团队还开发了新的计算模型以帮助分析光谱数据，包括卟啉复杂影响的一般理论模型血红素的电子滥用光谱上的荷花。该研究计划的结果使基本的血红素社区，通过阐明一种新颖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秒的影响来实现的在MHUD和ISDG活动位点中的球体相互作用在结构，电子结构和反应性上非经典血红素加氧酶催化的血红素降解的两个关键中间体。第一个目的研究团队将由于积极的站点变化而识别MHUD和ISDG的变体，功能变化。这 AIM是通过采用光谱镜来确定功能变化的变体来实现的，并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这些第二球如何功能变化和未改变的二级结构的变体扰动铁 - （氢）peroxo中间体。通过使用光谱法来快速识别具有扰动底物的变体来实现此目标电子结构，并采用磁光谱和理论计算来详细介绍电子结构发生变化及其对血红素 - 氧合反应性的影响。最后，研究的第三个目标团队将阐明MHUD和ISDG活性位点如何调整中羟基甲基的反应性。这研究小组员工空气敏感设备为这些反应性中间体准备光谱和机械表征。最终，该研究计划使用生化，光谱和计算工具来表征由非典型血红素氧化酶催化的两个氧合反应。