MECHANISM OF FERRITIN FERROXIDATION AND MINERALIZATION

铁蛋白铁氧化和矿化机制

• 批准号: 6490265
• 负责人: Boi-Hanh V. Huynh
• 金额: $ 18.75万
• 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 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6490265
• 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，几乎每一个方面都是必需的。 细胞功能。然而，在生理pH和氧化作用下 在条件下，它不是很容易溶解。生物是如何隔离铁的 因此，细胞利用率是一个至关重要的根本问题 重要性。此外，在存在02的情况下，自由Fe2+离子非常 有毒的，能够产生过氧化氢、超氧化物和其他 可攻击和破坏重要细胞的活性氧物种 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Fe2+离子。 溶液，并通过将氧化的Fe3+离子存储在 它的内部蛋白质腔以亚铁水合物的形式存在。然而， 尽管铁蛋白的功能和几十年的研究很重要 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 岩心(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，部分原因是 以前研究中使用的方法要么是间接的，要么是缺乏的 监测复杂反应所需的光谱分辨率 由铁蛋白催化。在此应用程序中，我们建议使用 穆斯堡尔谱学与快速冷冻-快速 用猝灭动力学技术研究铁蛋白的作用机理 铁氧化和矿化作用。三种不同的重组 铁蛋白，青蛙H和M铁蛋白，以及大肠杆菌细菌铁蛋白， 都要接受检查。我们初步研究的结果 证明这种动力学/光谱相结合的方法提供了 获得动力学信息所需的时间分辨率和 所需光谱分辨率用于区分、定量和 表征了在海平面上升过程中产生的多种铁物种 铁氧化和矿化过程。其他互补性 光谱，如EPR、Endor、EXAFS和共振拉曼也将 用来获得关于这些反应的进一步结构信息 中间体。特定部位的突变体将被设计、生产和 为达到以下目的而进行动力学/光谱研究 定义了氧化铁酶的位置、铁的运输途径和 某些关键残基的功能作用。一系列双重混合 57Fe和56Fe的快速冷冻-淬火穆斯堡尔研究 同位素是特别设计用来解决有关 铁蛋白功能的动力学。详细的机械洞察力 铁蛋白铁氧合酶反应过程与矿核 预计这些拟议的研究将形成新的结构。

项目成果

Addition of oxygen to the diiron(II/II) cluster is the slowest step in formation of the tyrosyl radical in the W103Y variant of ribonucleotide reductase protein R2 from mouse.

在小鼠核糖核苷酸还原酶蛋白 R2 的 W103Y 变体中，向二铁 (II/II) 簇添加氧是形成酪氨酰自由基的最慢步骤。

• DOI: 10.1021/bi7003747
• 发表时间: 2007
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Yun,Danny;Saleh,Lana;Garcia-Serres,Ricardo;Chicalese,BrandonM;An,YoungH;Huynh,BoiHanh;BollingerJr,JMartin
• 通讯作者: BollingerJr,JMartin

Facile electron transfer during formation of cluster X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse.

X簇形成过程中的轻松电子转移以及X在小鼠核糖核苷酸还原酶蛋白R2中产生酪氨酰自由基的动力学能力。

• DOI: 10.1021/bi011797p
• 发表时间: 2002
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Yun,Danny;Krebs,Carsten;Gupta,GovindP;Iwig,DavidF;Huynh,BoiHanh;BollingerJr,JMartin
• 通讯作者: BollingerJr,JMartin