Post-Fixation Nitrogen Cycle Metalloenzymology

固定后氮循环金属酶学

• 批准号: 10241363
• 负责人: Kyle M Lancaster
• 金额: $ 32.52万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10241363
• 关键词: Ammonia; Area; Bacteria; Biochemical Pathway; Biochemistry; Biological; Biology; Cardiac; Catalysis; Cell Respiration; Chemicals; Complement; Cytochromes; Development; Dioxygen; Dissociation; Electron Spin Resonance Spectroscopy; Electron Transport; Electrons; Environment; Enzymes; Goals; Health; Human; Hydroxylamine; Interdisciplinary Study; Iron; Ligands; Mediating; Membrane; Metals; Mission; Molecular; Molecular Biology; Molecular Structure; National Institute of General Medical Sciences; Nature; Nitrates; Nitric Oxide; Nitrites; Nitrogen; Nitrogen Dioxide; Nitrogen Fixation; Oxidants; Oxidases; Oxidation-Reduction; Oxidoreductase; Pathology; Play; Proteins; Protons; Reaction; Reactive Nitrogen Species; Reporting; Research; Role; Signal Transduction; Spectrum Analysis; Stimulus; Structure; Sulfur; System; Transition Elements; Vasodilation; Work; X ray spectroscopy; X-Ray Crystallography; biophysical properties; human pathogen; insight; iron nitrosyl; macromolecule; metalloenzyme; nitrification; novel therapeutic intervention; oxidation; programs; quantum; response; tool

Post-Fixation Nitrogen Cycle Metalloenzymology The long-term goal of the PI's research program is to understand how biology uses transition metals to control the speciation of redox-active substrates including reactive or “fixed” nitrogen species. Reactive nitrogen species serve vital roles in biology. For example, nitric oxide (NO) is a cellular signaling agent that regulates vasodilation in mammalian systems. In a separate context, nitrate (NO3–) can substitute for dioxygen (O2) as the terminal electron acceptor during cellular respiration by bacteria that include human pathogens. Of the biochemical pathways that generate these and other reactive nitrogen species, those involving oxidations of nitrogenous substrates are largely uncharacterized at the molecular level. This proposal describes an interdisciplinary research program leveraging molecular biology, biochemistry, inorganic spectroscopy, and quantum chemical calculations to understand in precise detail the mechanisms used by metalloenzymes operative in nitrification––biological ammonia (NH3) and nitrite (NO2–) oxidation. Despite the global scale on which this biochemistry operates and the impacts of nitrification products on the environment and on human health, detailed mechanisms for the operative metalloenzymes are unavailable. Early work from the PI has afforded a revised mechanism used by the nitrification enzyme cytochrome P460 for the step-wise, selective oxidation of hydroxylamine (NH2OH), an intermediate in NH3 oxidation. A key iron-nitrosyl (FeNO) intermediate has since been identified that gates catalysis via axial ligand dissociation. The structural and electronic factors that dictate the conversion between catalytically competent and incompetent forms will be explored to gain insight into similar mechanisms operative in NO-mediated cellular signaling. These studies will directly probe metal-NO bonding using resonance Raman, electron paramagnetic resonance, and X-ray spectroscopy. Work on biological NH2OH oxidation will be extended to previously reported but largely uncharacterized non-heme Fe hydroxylamine oxidases. Understanding of NH2OH-oxidation mechanisms will elucidate means by which nature mediates the intermediacy of a toxic metabolite during cellular energy transduction. A full suite of biophysical characterization will be carried out including X-ray crystallography and Fe-focused spectroscopy. Finally, the mechanism of NO2– oxidation by the integral membrane metalloenzymes nitrite oxidoreductase (NXR) will be studied. No molecular structure is available for NXR, and its complement of metallocofactors is undefined. NXR is thought to mediate multi-electron transfer using multiple iron-sulfur clusters. Spectroelectrochemical probing of NXR will be carried out to afford new understanding of how nature controls multi-step electron flow using chains of metallocofactors. This program proposal offers progress in a number of NIGMS mission areas including the development of spectroscopic tools, the study of biosyntheses of cellular signaling agents, and the study of biological electron transport involving the management of multiple protons and electrons.

固氮后氮循环金属酶学 PI研究计划的长期目标是了解生物学如何利用过渡金属来 控制氧化还原活性底物的物种形成，包括活性或“固定”氮物种。反应性 氮物种在生物学中起着重要作用。例如，一氧化氮（NO）是细胞信号传导剂， 调节哺乳动物系统中的血管舒张。在一个单独的背景下，硝酸盐（NO3-）可以取代分子氧 (O2)作为包括人类病原体在内的细菌的细胞呼吸过程中的终端电子受体。的 产生这些和其他活性氮物质的生化途径，包括氧化 的含氮底物在很大程度上是在分子水平上的特点。该提案描述了一个 利用分子生物学、生物化学、无机光谱学和 量子化学计算，以精确了解金属酶使用的机制 在硝化-生物氨（NH3）和亚硝酸盐（NO2-）氧化中起作用。尽管全球范围内， 硝化产物对环境和人类的影响 健康，详细的机制，工作金属酶是不可用的。PI的早期工作 提供了一个修订的机制所使用的硝化酶细胞色素P460的逐步，选择性 羟胺（NH 2 OH）的氧化，羟胺是NH3氧化的中间体。一种关键的铁-亚硝酰（FeNO）中间体 已经被鉴定为通过轴向配体解离进行门控催化。结构和电子因素 决定催化能力和不称职的形式之间的转换将被探索，以获得 深入了解NO介导的细胞信号传导中的类似机制。这些研究将直接探讨 使用共振拉曼、电子顺磁共振和X射线光谱法的金属-NO键合。工作 生物NH 2 OH氧化将扩展到以前报道的，但很大程度上未表征的非血红素 铁羟胺氧化酶。了解NH 2 OH氧化机制将阐明 自然界在细胞能量转导过程中介导毒性代谢物的中间性。一整套 将进行生物物理表征，包括X射线晶体学和铁聚焦光谱学。 最后，NO2-氧化的机制，由整合膜金属酶亚硝酸盐氧化还原酶 (NXR)将被研究。NXR没有分子结构，其金属辅因子的补充是 未定义。NXR被认为是使用多个铁硫簇介导多电子转移。 NXR的光谱电化学探测将被进行，以提供对自然如何控制的新理解。 使用金属辅因子链的多步电子流。该计划提案在一些方面取得了进展， NIGMS的使命领域包括光谱工具的开发，细胞生物合成的研究， 信号传导剂，以及涉及多个质子管理的生物电子传递的研究 和电子。