Iron Deposition and Mobilization in Ferritin

描述(申请人提供)：从微生物到人类，铁蛋白在铁的生物管理中发挥着核心作用。铁蛋白通过在其壳状结构中以水合三氧化二铁矿物的形式沉积铁，起到铁储存和解毒蛋白的作用。这种铁随后可以被动员起来，用于合成血红素。尽管来自不同生物体的铁蛋白有许多共同的结构特征，无论是12亚基还是24亚基蛋白质，但它们在铁沉积的化学成分上存在显著差异。所有已知的铁蛋白都含有催化分子氧或过氧化氢氧化铁(II)的铁氧合酶；然而，它们以显着不同的方式做到这一点。本研究重点研究了多种重组24聚铁蛋白的铁沉积机制，其中包括人H链和L链铁蛋白、一种新发现的人线粒体铁蛋白、含血红素的大肠杆菌细菌铁蛋白(EcBFR)和大肠杆菌诺氏细菌铁蛋白(EcFtnA)。还将对12聚体蛋白：无核李斯特菌铁蛋白和来自大肠杆菌的DNA结合蛋白DPS进行研究。对于不同的蛋白质，将讨论有关氧结合、铁氧化过程中铁蛋白-铁蛋白结合、瞬时自由基和铁中间体以及以氧气和过氧化氢为氧化剂的铁氧化和铁水解的化学计量方程等重要问题。通过定点突变结合X射线结构数据、等温滴定量热法、紫外可见停流动力学、快速冷冻猝灭穆斯堡尔谱和EPR谱、自旋捕捉、光散射、血氧仪和pH恒定，将阐明各种铁蛋白的铁沉积机制的特征。所提出的广泛研究将导致详细了解各种铁蛋白如何作为可逆的铁储存蛋白发挥作用，帮助细胞应对氧化应激，并促进我们对铁生物矿化过程的一般化学和生物化学的了解。

DESCRIPTION (provided by applicant): From microorganisms to mankind, ferritin plays a central role in the biological management of iron. The ferritins function as iron storage and detoxification proteins by depositing iron as a hydrous ferric oxide mineral within their shell-like structures. This iron can be subsequently mobilized for the synthesis of heme. While ferritins from various organisms share many common structural features, being either 12 or 24 subunit proteins, they differ markedly in their chemistries of iron deposition. All known ferritins contain ferroxidase sites that catalyze iron(II) oxidation by either molecular oxygen or hydrogen peroxide; however they do so in significantly different ways. This proposal focuses on the mechanisms of iron deposition in a variety of recombinant 24mer ferritins that include human H- and L-chain ferritins, a newly discovered human mitochondrial ferritin, the heme-containing E. coli bacterioferritin (EcBFR) and the E. coil northeme bacterial ferritin (EcFtnA). Studies will also be conducted with the l2mer proteins: Listeria innocua ferritin and the DNA binding protein, Dps from E. coli. Important questions relating to dioxygen binding, ferritin-ferritin association during iron oxidation, transient radical and iron intermediates and the stoichiometric equations for iron oxidation and hydrolysis using dioxygen and hydrogen peroxide as oxidants will be addressed for the different proteins. Features of the mechanisms of iron deposition of the various ferritins will be elucidated through a combination of site-directed mutagenesis in conjunction with x-ray structure data, isothermal titration calorimetry, UV-visible stopped-flow kinetics, rapid-freeze quench Mossbauer and EPR spectroscopies, spin trapping, light scattering, oximetry and pH stat. The extensive studies proposed should lead to a detailed understanding of how various ferritins function as reversible iron storage proteins, assisting the cell cope with oxidative stress, and further our knowledge of the chemistry and biochemistry of iron biomineralization processes in general.