MECHANISM OF FERRITIN FERROXIDATION AND MINERALIZATION

铁蛋白铁氧化和矿化机制

• 批准号: 6343059
• 负责人: Boi-Hanh V. Huynh
• 金额: $ 18.4万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6343059
• 关键词: Mossbauer spectrometry; Raman spectrometry; bacterial proteins; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; ferritin; iron; mutant; oxidation; protein structure function; recombinant proteins; ultraviolet spectrometry

Iron is an important nutrient, required in almost every aspect of cellular function. However, at physiological pH and under oxidizing condition, it is not very soluble. How living organisms sequester iron for cellular utilization is therefore a fundamental question of vital importance. Also, in the presence Of 02, free Fe2+ ions are extremely toxic, capable of generating hydrogen peroxide, superoxide, and other reactive oxygen species that can attack and destroy important cellular molecules. Ferritin is unique in the sense that it performs dual functions of iron detoxification, by oxidizing the Fe2+ ions in solution, and iron sequestration, by storing the oxidized Fe3+ ions in its inner protein cavity in the form of ferrihydrite mineral. However, despite the importance of ferritin functions and decades of research efforts, the mechanism by which ferritin catalyzes the Fe2+ oxidation (ferroxidation) and directs the oxidized products to form the mineral core (mineralization) is still poorly understood. This is partly due to the complexity of the ferritin molecule and partly due to the fact that the methods used in previous studies were either indirect or lacked the required spectroscopic resolution to monitor the complex reaction catalyzed by ferritin. In this application, we propose to employ Mssbauer spectroscopy in conjunction with the rapid freeze-rapid quench kinetic technique to investigate the mechanism of ferritin ferroxidation and mineralization. Three different recombinant ferritins, the frog H and M ferritins, and the E. coli bacterioferritin, are to be examined. Results obtained from our preliminary studies demonstrate that this combined kinetic/spectroscopic approach provides the necessary time resolution for obtaining kinetic information and the required spectroscopic resolution for distinguishing, quantifying and characterizing the multiple Fe species generated during the ferroxidation and mineralization processes. Other complementary spectroscopies, such as EPR, ENDOR, EXAFS, and resonance Raman will also be employed to obtain further structural information on these reaction intermediates. Site-specific mutants will be engineered, produced and subjected to kinetic/spectroscopic investigations for the purpose of defining the ferroxidase site, the Fe transport pathways, and the functional roles of certain key residues. A series of double-mixing rapid freeze-quench Mossbauer investigations using 57Fe and 56Fe isotopes are particularly designed to address questions concerning the dynamics of the ferritin function. Detailed mechanistic insights into the processes involved in ferritin ferroxidase reaction and mineral core formation are expected to emerge from these proposed studies.

铁是一种重要的营养素，几乎各个方面都需要铁 细胞功能。 然而，在生理pH值和氧化条件下 条件下，它不太溶解。 生物体如何吸收铁 因此，细胞利用是一个至关重要的基本问题 重要性。 此外，在存在 02 的情况下，游离 Fe2+ 离子极其 有毒，能产生过氧化氢、超氧化物等 活性氧可以攻击和破坏重要的细胞 分子。铁蛋白的独特之处在于它具有双重功能 铁的解毒功能，通过氧化铁中的 Fe2+ 离子 溶液和铁螯合，通过将氧化的 Fe3+ 离子储存在 其内部蛋白质空腔以水铁矿形式存在。然而， 尽管铁蛋白功能很重要并且经过了数十年的研究 铁蛋白催化Fe2+氧化的机制 （铁氧化）并引导氧化产物形成矿物质 核心（矿化）仍然知之甚少。 这部分是由于 由于铁蛋白分子的复杂性，部分原因在于 以前的研究中使用的方法要么是间接的，要么缺乏 监测复杂反应所需的光谱分辨率 由铁蛋白催化。在此应用中，我们建议采用 穆斯堡尔谱与快速冷冻-快速 淬灭动力学技术研究铁蛋白的机制 铁氧化和矿化。 三种不同的重组体 铁蛋白、青蛙 H 和 M 铁蛋白以及大肠杆菌铁蛋白， 都需要被检查。 我们的初步研究得出的结果 证明这种结合的动力学/光谱方法提供了 获得动力学信息所需的时间分辨率和 区分、量化和分析所需的光谱分辨率 表征过程中产生的多种铁形态 铁氧化和矿化过程。 其他补充 光谱学，例如 EPR、ENDOR、EXAFS 和共振拉曼也将 用于获得有关这些反应的进一步结构信息 中间体。 位点特异性突变体将被设计、生产和 进行动力学/光谱研究，目的是 定义亚铁氧化酶位点、铁转运途径和 某些关键残基的功能作用。 系列双混 使用 57Fe 和 56Fe 进行快速冷冻淬灭穆斯堡尔研究 同位素是专门为解决有关 铁蛋白功能的动态变化。 详细的机制见解 铁蛋白亚铁氧化酶反应和矿物质核心涉及的过程 预计这些拟议研究将形成。