MECHANISM OF FERRITIN FERROXIDATION AND MINERALIZATION

铁蛋白铁氧化和矿化机制

• 批准号: 6138700
• 负责人: Boi-Hanh V. Huynh
• 金额: $ 17.96万
• 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/6138700
• 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.

铁是重要的营养素，几乎需要 细胞功能。 但是，在生理pH和氧化下 条件，它不是很可溶。 生物如何隔离铁 因此，对于蜂窝使用是至关重要的基本问题 重要性。 同样，在02存在的情况下，Free Fe2+离子极为 有毒，能够产生过氧化氢，超氧化物和其他 活性氧，可以攻击并破坏重要的细胞 分子。铁蛋白是独一无二的 铁解毒的功能，通过氧化Fe2+离子中的功能 溶液和铁固存，将氧化的Fe3+离子存储在 其内部蛋白质腔以铁硫酸盐矿物质的形式。然而， 尽管铁蛋白功能和数十年的研究很重要 努力，铁蛋白催化Fe2+氧化的机制 （铁氧化）并指示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 达到铁蛋白分子的复杂性，部分原因是 以前研究中使用的方法是间接或缺乏 所需的光谱分辨率以监测复杂反应 铁蛋白催化。在此应用程序中，我们建议雇用 M SSBAUER光谱与快速冻结相结合 淬灭动力学技术来研究铁蛋白的机理 铁氧化和矿化。 三个不同的重组 铁蛋白，青蛙H和M铁蛋白以及大肠杆菌细菌铁蛋白， 要检查。 从我们的初步研究获得的结果 证明这种组合的动力学/光谱方法提供了 获得动力学信息的必要时间分辨率和 所需的光谱分辨率，以区分，量化和 表征在 铁氧化和矿化过程。 其他补充 EPR，ENDOR，EXAFS和共振拉曼等光谱镜也将 被用来获得有关这些反应的进一步结构信息 中间人。 现场特异性突变体将进行设计，生产和 进行动力学/光谱研究 定义铁氧化酶部位，FE传输途径和 某些关键残基的功能作用。 一系列双重混合 使用57FE和56FE进行快速冻结摩斯鲍尔调查 同位素的设计旨在解决有关的问题 铁蛋白功能的动力学。 详细的机械洞察力 涉及铁蛋白铁氧化酶反应和矿物核的过程 预计从这些提出的研究中得出形成。