STRUCTURE AND FUNCTION OF FERROCHELATASE

这是一个香农奖提供部分支持的研究 项目不属于指定的研究所的资金范围，但 都处于优秀的边缘。 香农奖旨在 提供支持，以测试该方法的可行性;进一步发展 测试和完善研究技术;进行二次分析 可用的数据集;或进行离散项目， PI的研究能力或为已经存在的 有价值的申请。 下面的摘要摘自原文 主要研究者提交的文件。 血红素在细胞功能的几乎每个方面都是必不可少的，因为它 是蛋白质中必需的辅基， 功能协调发展的 铁螯合酶（原血红素铁裂解酶，EC 4.99.1.1）催化 亚铁离子插入原卟啉IX形成产物 血红素IX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， 铁螯合酶由于难以获得 足够量的纯化的活性酶，部分原因是 铁螯合酶与细胞膜的结合， 线粒体中的亚铁螯合酶。 我们大多数的知识， 铁螯合酶的活性位点来自动力学研究， 化学修饰的亚铁螯合酶和抑制研究。 更直接 物理研究，如光谱研究，是不可能的， 这是由于纯化蛋白的可用性有限。 这一重大障碍 被我们的合作者G博士克服了C.费雷拉，谁 成功地在大肠杆菌中过表达鼠亚铁螯合酶 杆菌 最重要的是，重组酶与 细菌可溶性级分，便于随后的纯化， 操纵酶。 我们现在有了基本上无限的 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表明， 重组酶揭示了重要和令人惊讶的结果。 也就是说， 哺乳动物亚铁螯合酶是一种含有[2Fe-2S] 集群 这些新的发展开辟了许多新的途径， 铁螯合酶研究。 因此，我们建议采用以下方法 光谱、化学、生物化学和分子生物学方法 研究铁螯合酶的结构和功能。 在 特别地，我们建议（1）应用穆斯堡尔谱、EPR谱和NMR谱 以表征和获得有关铁的结构信息， 结合位点和[2Fe-2S]簇，（2）鉴定氨基酸 使用定点的铁螯合酶功能必需的残基 诱变与光谱和动力学研究相结合， （3）用快速电泳技术阐明亚铁螯合酶的作用机理 冷冻淬火EPR和穆斯堡尔技术。 由于亚铁螯合酶使用 铁作为底物，我们的初步结果表明，它是 一种含铁的酶，提出的光谱方法， 穆斯堡尔和EPR技术的结合，特别适合于 铁螯合酶的研究。 预计拟议的调查将 以产生特征物理性质和结构信息 关于底物铁结合位点和2个[Fe-2S]簇，和 以提供详细的机械信息。 可能的反应 中间体可以被捕获和表征，其速率 将确定形成和衰变。 拟议的研究还 预期阐明[2Fe-2S]簇在 铁螯合酶，并提供新的见解，一般铁的作用 蛋白质中的硫簇。

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.