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，几乎在每个方面都需要。 细胞功能。 然而，在生理pH和氧化条件下， 条件下，它不是很可溶。 生物体如何螯合铁 因此，对于蜂窝利用来说， 重要性 此外，在O2的存在下，游离Fe 2+离子是极其不稳定的。 有毒，能够产生过氧化氢，超氧化物，和其他 活性氧可以攻击和破坏重要的细胞 分子。铁蛋白是独特的，因为它执行双重功能， 铁解毒功能，通过氧化铁离子中的Fe 2+离子， 溶液，和铁螯合，通过储存氧化的Fe 3+离子， 其内部蛋白质腔以水铁矿矿物的形式存在。然而， 尽管铁蛋白功能的重要性和数十年的研究 铁蛋白催化Fe 2+氧化的机制 （铁氧化）并引导氧化产物形成矿物 核心（矿化）仍然知之甚少。 这部分是由于 铁蛋白分子的复杂性，部分原因是 以前的研究中使用的方法要么是间接的， 监测复杂反应所需的光谱分辨率 由铁蛋白催化在本申请中，我们建议采用 M穆斯堡尔谱结合快速冷冻-快速 淬灭动力学技术研究铁蛋白的作用机理 铁氧化成矿作用 三种不同的重组 铁蛋白、蛙H和M铁蛋白以及E.大肠杆菌细菌铁蛋白， 都要接受检查 我们初步研究的结果 证明这种结合动力学/光谱方法提供了 获得动力学信息所需的时间分辨率， 所需的光谱分辨率，用于区分，定量和 表征了在反应过程中产生的多种Fe物质， 铁氧化和矿化过程。 其他互补 光谱学，如EPR、ENDOR、EXAFS和共振拉曼，也将 用于获得这些反应的进一步结构信息 中间体的 位点特异性突变体将被工程化，生产和 进行动力学/光谱研究，目的是 确定铁氧化酶位点、铁转运途径和铁转运途径。 某些关键残基的功能作用。 一系列双混频 用~（57）Fe和~（56）Fe研究快速冷冻淬火穆斯堡尔谱 同位素是特别设计来解决有关的问题， 铁蛋白功能的动力学。 详细的机械见解， 参与铁蛋白铁氧化酶反应和矿物核的过程 预计这些拟议的研究将产生新的结构。

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