Post-Fixation Nitrogen Cycle Metalloenzymology

固定后氮循环金属酶学研究 PI研究计划的长期目标是了解生物如何利用过渡金属 控制氧化还原活性底物的形态，包括活性或固定的氮物种。反应性 氮物种在生物学中起着至关重要的作用。例如，一氧化氮(NO)是一种细胞信号转导因子， 调节哺乳动物系统中的血管扩张。在另一种情况下，硝酸盐(NO3-)可以替代氧气 (O2)作为包括人类病原体在内的细菌在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种的生化途径，那些涉及氧化的 在分子水平上很大程度上没有表征含氮底物。本提案描述了一种 利用分子生物学、生物化学、无机光谱学和 用量子化学计算精确详细地了解金属酶的作用机制 用于硝化--生物氨(NH3)和亚硝酸盐(NO2-)氧化。尽管全球范围内 以及硝化产物对环境和人类的影响 健康方面，目前尚无金属酶起作用的详细机制。PI的早期工作有 提供了一个由硝化酶细胞色素p460用于分步的、选择性的 羟胺的氧化，这是NH3氧化的中间产物。一种关键的亚硝酸铁(FeNO)中间体 自那以后，人们发现GATES通过轴向配体解离来催化。结构和电子因素 决定了催化活性形式和非催化活性形式之间的转换，将被探索以获得 深入了解在NO介导的细胞信号中起作用的类似机制。这些研究将直接探索 金属-使用共振拉曼、电子顺磁共振和X射线光谱分析进行无键连接。工作 关于生物的NH_2OH氧化将扩展到以前报道的但基本上未描述的非血红素 铁羟胺氧化酶。对NH_2OH氧化机理的理解将阐明 在细胞能量转导过程中，自然界对有毒代谢物的中介起着中介作用。全套的 将进行生物物理表征，包括X射线结晶学和铁聚焦光谱分析。 最后，探讨了完整膜金属酶亚硝酸氧化还原酶氧化NO2-的机理。 (NXR)将被研究。NXR没有可用的分子结构，它的金属辅因子是 未定义。NXR被认为通过多个铁-硫团簇来调节多电子转移。 将对NXR进行光谱电化学探测，以提供对自然控制的新理解 使用金属辅因子链的多步骤电子流动。该计划提案在许多方面取得了进展 NIGMS的任务领域包括光谱工具的开发，细胞生物合成的研究 与多质子管理有关的生物电子传输的研究 和电子。

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.