Proton Translocation and Metal Chelation in Ferrochelatase

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。智力MeritHeme是一种重要的辅助因子，用于多种生物学上重要的蛋白质，包括球蛋白，细胞色素，转录因子，过氧化物酶，过氧化氢酶等。除了少数例外，具有含血红素蛋白质的生物体具有合成自身血红素的生物合成机制，并且不从其饮食中获得血红素。不能合成血红素对大多数生物来说是致命的。血红素的代谢合成的重要性是强调最近的证据表明，血红素也是许多其他代谢过程的调节剂。在这个令人兴奋的新角色中，这些“血红素传感器”必须检测和响应血红素，但不能永久地结合它。七步生物合成途径中的最后一步，亚铁插入原卟啉以产生血红素（血红素），由亚铁螯合酶催化。在动物中，这种膜相关酶位于线粒体内膜的基质侧，其活性位点面向膜。这与非常可溶的细菌亚铁螯合酶相反。 这项研究的核心是开发生物物理工具和技术来直接监测卟啉大环中的质子易位以及金属结合和螯合。 这种技术和实验发现的影响具有更广泛的影响，因为质子结合/转移是绝大多数生物化学反应和许多基本生物化学过程的关键要素。 此外，现在人们普遍认识到，金属在所有已知酶促反应中约30%起着关键作用。 利用自然界中发现的催化多样性将是至关重要的，以维持人类目前享有的生活质量，在缺乏丰富的化石燃料。由于螯合反应在这里调查的广泛重要性，在这个项目中概述的实验将显着推进我们的理解金属是如何插入到卟啉辅因子一般。此外，该项目的一个独特的方面是有机合成和中子衍射的应用，以测试有关卟啉去质子化，金属脱水和金属插入的原子细节的具体假设。鉴于卟啉辅因子在许多酶、代谢途径以及临床应用中的既定和新兴作用，这项工作将具有深远的意义。此外，中子衍射方法和有机合成在探测酶机制方面的应用使该项目真正具有多学科性，并将这项工作置于科学发现的前沿，这将导致并支持新技术的发展。这个研究项目提供了一个特殊的教育机会，在多学科的方法来推进发现和结构的理解的背景下，酶的功能关系。资金将用于支持在生物物理化学、结构生物学、生物化学、分子生物学和有机化学方面培训两名博士候选人和几名本科生。此外，该项目将通过与Peach State Louis Stokes少数民族参与联盟（LSAMP）以及本科生研究机会中心（CURO）计划的联系，进一步促进对物理科学的吸引力。 这两个项目在帮助这些学生在他们选择的专业领域取得成功方面都有良好的记录。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Intellectual MeritHeme is an essential cofactor for a wide variety of biologically important proteins including globins, cytochromes, transcription factors, peroxidases, catalases, and others. With few exceptions, organisms that possess heme-containing proteins possess the biosynthetic machinery to synthesize their own heme and do not acquire heme from their diet. Inability to synthesize heme is lethal for most organisms. The importance of the metabolic synthesis of heme is underscored by recent evidence revealing that heme is also a regulator of a number of other metabolic processes. In this exciting new role, these "heme sensors" must detect and respond to heme but not bind it permanently. The terminal step in the seven step biosynthetic pathway, the insertion of ferrous iron into protoporphyrin to make protoheme (heme), is catalyzed by the enzyme ferrochelatase. In animals this membrane-associated enzyme is located on the matrix side of the inner mitochondrial membrane with its active site facing into the membrane. This is in contrast to the bacterial ferrochelatases that are quite soluble. At the heart of this investigation is the development of biophysical tools and techniques to directly monitor proton translocation as well as metal binding and chelation in porphyrin macrocycles. The implications of such technology and experimental findings have much broader implications because proton binding/transfer is a critical element of the vast majority of biochemical reactions and many essential biochemical processes. In addition, it is now widely recognized that metals play a critical role in approximately 30 percent of all known enzymatic reactions. Taking advantage of the catalytic diversity found in nature will be critical to sustaining the quality of life humanity currently enjoys in the absence of abundant fossil fuels.Due to the broad importance of the chelation reaction under investigation here, the experiments outlined in this project will significantly advance our understanding of how metals are inserted into porphyrin cofactors in general. In addition, a unique aspect of this project is the application of organic synthesis and neutron diffraction to test specific hypotheses regarding the atomic details of porphyrin deprotonation, metal dehydration, and metal insertion. Given the established and emerging roles for porphyrin cofactors in numerous enzymes, metabolic pathways, as well as clinical applications, this work will have far reaching implications. In addition, the application of both neutron diffraction methods and organic synthesis to probe enzyme mechanism makes this project truly multidisciplinary and places this work at the cutting edge of scientific discovery that will lead to and support new technology development.Broader ImpactIn addition to the broader technological impacts, this research project offers an exceptional educational opportunity in the context of a multi-disciplinary approach to advancing discovery and understanding of structure-function relationships in enzyme function. Funding will support training of two PhD candidates and several undergraduate students in biophysical chemistry, structural biology, biochemistry, molecular biology and organic chemistry. In addition, this project will further advance the attraction of underrepresented minority students to the physical sciences through an affiliation with the Peach State Louis Stokes Alliance for Minority Participation (LSAMP) as well as the Center for Undergraduate Research Opportunities (CURO) programs. Both of these programs have proven track records in helping these students find success in the professional fields of their choice.