STRUCTURE AND FUNCTION OF FERROCHELATASE

铁螯合酶的结构和功能

• 批准号: 2190797
• 负责人: Boi-Hanh V. Huynh
• 金额: $ 10万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 1998-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2190797
• 关键词: active sites; lyase; metalloenzyme; protein structure function

This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. Heme is essential in almost every single aspect of cell function, as it is a required prosthetic group in proteins with very diversified functions. Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) catalyzes the insertion of ferrous ion into protoporphyrin IX forming the product protoheme IX and has been recognized as a key enzyme in heme biosynthesis since 1956. Unfortunately, for the past decades, research on ferrochelatase has been hindered by the difficultly in obtaining sufficient quantities of purified active enzyme, in part due to the association of ferrochelatase with membranes and the low abundance of ferrochelatase in mitochondria. Most of our knowledge concerning the active sites of ferrochelatase was derived from kinetic studies of chemically modified ferrochelatase and inhibition studies. More direct physical investigations, such as spectroscopic studies, were not possible due to the limited availability of purified proteins. This major hurdle has been overcome by one of our collaborators, Dr. G. C. Ferreira, who has successfully overexpressed the murine ferrochelatase in Escherichia coli. Most importantly, the recombinant enzyme is associated with the bacterial soluble fraction, facilitating the subsequent purification and manipulation of the enzyme. We now have essentially unlimited quantities of purified active ferrochelatase for spectroscopic and kinetic studies. Our preliminary EPR and Mossbauer spectroscopic studies of the recombinant enzyme reveal an important and surprising result. That is, mammalian ferrochelatase is a metalloenzyme containing a [2Fe-2S] cluster. These new developments have opened up many new avenues for ferrochelatase research. We therefore propose to use a combination of spectroscopic, chemical, biochemical, and molecular biological approaches to investigate the structure and function of ferrochelatase. In particular, we propose (1) to apply Mossbauer, EPR and NMR spectroscopy to characterize and to obtain structural information about the ferrous binding site and the [2Fe-2S] cluster, (2) to identify the amino-acid residues essential for ferrochelatase function using site-directed mutagenesis in combination with spectroscopic and kinetic investigations, and (3) to elucidate the mechanism of ferrochelatase employing rapid freeze-quench EPR and Mossbauer techniques. Since ferrochelatase uses iron as a substrate and our preliminary results established that it is an iron-containing enzyme, the proposed spectroscopic approach, a combination of Mossbauer and EPR techniques, is particularly suited for the studies of ferrochelatase. The proposed investigations are expected to yield characteristic physical properties and structural information concerning the substrate iron binding site and the 2[Fe-2S] clusters, and to provide detailed mechanistic information. Possible reaction intermediates may be trapped and characterized, and its rates of formation and decay will be determined. the proposed studies are also expected to shed light on the functional role of the [2Fe-2S] cluster in ferrochelatase and provide new insights in general into the role of iron sulfur clusters in proteins.

这是香农奖，为这项研究提供部分支持 项目低于指定研究所的资助范围，但 都处于最优秀的边缘。香农奖旨在 为测试该方法的可行性提供支持；进一步发展 测试和改进研究技术；执行二次分析 可用的数据集；或执行可演示的离散项目 PI的研究能力或为已经 值得称道的申请。以下摘要摘自原文 首席调查员提交的文件。 血红素几乎在细胞功能的每一个方面都是必不可少的，因为它 是蛋白质中一个必需的修复基团，具有非常多样化的 功能。铁络合酶(原血红素铁水解酶，EC 4.99.1.1)催化 原卟啉IX中亚铁离子的插入反应 原血红素IX，已被认为是血红素生物合成的关键酶 从1956年开始。不幸的是，在过去的几十年里，对 铁络合酶由于难以获得而受到阻碍 足够数量的纯化活性酶，部分原因是 铁络合酶与膜的结合及低丰度 线粒体中的铁络合酶。我们所知道的大部分关于 铁络合酶的活性部位是通过动力学研究得出的 化学修饰的铁络合酶及其抑制研究。更直接 物理研究，如光谱研究，是不可能的。 由于纯化蛋白的可获得性有限。这一主要障碍 已经被我们的一位合作者G.C.费雷拉博士克服了，他 已成功在大肠杆菌中过表达鼠铁络合酶 Coli.最重要的是，重组酶与 细菌可溶部分，便于后续的纯化和 对酶的操纵。我们现在基本上有无限的数量 纯化的活性铁络合酶，用于光谱和动力学研究。 我们初步的EPR和穆斯堡尔谱学研究 重组酶揭示了一个重要而令人惊讶的结果。那是, 哺乳动物铁络合酶是一种含有[2Fe-2S]的金属酶 集群。这些新的发展开辟了许多新的途径 铁络合酶研究。因此，我们建议结合使用 光谱、化学、生化和分子生物学方法 研究铁络合酶的结构和功能。在……里面 具体地说，我们建议(1)应用穆斯堡尔谱、EPR谱和核磁共振谱 以表征和获取有关铁的结构信息 结合部位和[2Fe-2S]簇，(2)鉴定氨基酸 利用定点定位技术实现铁络合酶功能所必需的残基 结合光谱和动力学研究的诱变， (3)利用RAPID阐明铁络合酶的作用机制 冷冻淬火EPR和穆斯堡尔技术。由于络合铁酶使用 铁作为底物，我们的初步结果证实了它是 一种含铁酶，建议的光谱方法，一种 穆斯堡尔和EPR技术的结合，特别适合于 铁络合酶的研究。预计将进行拟议的调查 以获得特定的物理属性和结构信息 关于底物铁结合位置和2[Fe-2S]簇，以及 提供详细的机械信息。可能的反应 中间体可能会被捕获和表征，其速率 形成和腐烂将被确定。拟议的研究还包括 预计将阐明[2Fe-2S]团簇在 并对铁的作用提供了新的见解 蛋白质中的硫簇。