Understanding the molecular mechanism of iron sulfur cluster biogenesis

电池的工作得益于从环境中吸收的“燃料”。由铁和硫组成的小分子（称为铁硫簇）附着在蛋白质上，在将食物（或光）中储存的能量转化为呼吸（和光合作用）中可用的形式的过程中起着关键作用。然而，这些铁硫簇分子并不能自发形成，需要直接在细胞中组装。这一过程带来了一个重要的问题：铁和硫是生命所必需的元素，但同时也具有内在的毒性。因此，大自然设计了非常复杂和严格调控的分子机器，在生命王国中进化保守，并且在过去的15-20年中发现了所有这些分子机器，以合成铁硫簇分子，并以非常有序和受调控的方式将它们附着在蛋白质上，确保最大限度地减少浪费和对细胞的潜在伤害。这些机器对人类生活的重要性由越来越多的疾病的数量来说明，这些疾病似乎与铁硫簇蛋白的损伤及其形成有关。当这些机器的任何一个部分发生故障时，疾病就会发生。我们过去的研究工作主要集中在理解这个重要问题和铁硫簇生物化学上，产生了几篇开创性的论文，大大推进了这一领域。对人类和细菌的铁硫簇组装机器的日益复杂的理解正在慢慢出现。然而，我们仍然远远没有得到全貌，并且由于缺乏关于事件的精确顺序和中间体的性质的详细信息，我们提出了一个项目，旨在了解前所未有的详细机制，该机制是由铁和半胱氨酸形成的（硫的来源）。具体而言，试验方案将解决关键的未决问题，包括：铁和硫传递的精确机制，导致簇形成的步骤，共济失调蛋白的精确作用，（一种与人类遗传疾病弗里德赖希共济失调有关的蛋白质，已知是铁硫簇组装的重要组成部分），将簇转移到需要簇发挥功能的载体和/或靶蛋白的过程。我们将使用不同的生物物理，结构和生物化学技术的强大组合，这将使我们能够重建整个机制。我们已经开发了所有建议技术的所有必要知识和专业知识。特别新奇的是在蛋白质保持折叠的条件下将质谱法应用于铁硫簇蛋白。这具有巨大的优点，即铁-硫簇及其片段保持与折叠的蛋白质结合，使得通过精确测量结合有辅因子的蛋白质的质量，可以推断出辅因子的身份。最近，这提供了前所未有的洞察铁硫簇的转换和降解在其他系统中，是一个非常有前途的，新的方法来阐明从头簇assembly.Overall的步骤，我们的研究有重要意义，我们的基本理解这些细胞过程中的细菌，这是在很大程度上保存在人类，具有潜在的长期医疗效益。

Cells work thanks to 'fuel' which is absorbed from the environment. Small molecules made of iron and sulfur (called iron-sulfur clusters), which are attached to proteins, play key roles in the process by which energy stored in food (or light) is converted to a useable form in respiration (and photosynthesis). These iron-sulfur cluster molecules do not form spontaneously, however, and need to be assembled directly in the cell. This process poses an important problem: iron and sulfur are elements which are essential for life but, at the same time, also intrinsically toxic. Nature has thus engineered very complex and tightly regulated molecular machines, evolutionarily conserved across the kingdoms of life and all discovered within the past 15-20 years, to synthesise iron-sulfur cluster molecules and attach them to proteins in a very orderly and regulated way, making sure to minimise waste and the potential for harm to the cell. The importance of these machines for human life is illustrated by the number of diseases which increasingly appear to be linked to impairment of iron-sulfur cluster proteins and their formation. When any of the parts of these machines break down, disease occurs.We have in the past focused a major part of our research efforts on understanding this important problem and on iron-sulfur cluster biochemistry in general, resulting in several seminal papers which have substantially advanced the field. An increasingly sophisticated understanding of the iron-sulfur cluster assembly machines of humans and bacteria is slowly emerging. However, we are still far from having the full picture, and understanding the steps involved in formation of the cluster is limited by the lack of detailed information on the precise sequence of events and nature of intermediates, which interactions are formed and how they regulate this process.We propose a project aimed at understanding in unprecedented detail the mechanism by which the cluster is formed from iron and cysteine (the source of sulfur). Specifically, the experimental programme will address crucial outstanding questions including: the precise mechanism by which iron and sulfur are delivered, the steps that lead to cluster formation, the precise role of frataxin (a protein linked to the genetic disease Friedreich's ataxia in humans and know to be an important component of iron-sulfur cluster assembly), the process of cluster transfer to carrier and/or target proteins that require a cluster for function. We will use a powerful combination of different biophysical, structural and biochemical techniques which will allow us to reconstruct the whole mechanism. We have already developed all the necessary know-how and expertise in all the proposed techniques. Of particular novelty is the application of mass spectrometry to iron-sulfur cluster proteins under conditions in which the protein remains folded. This has the tremendous advantage that iron-sulfur clusters, and fragments thereof, remain bound to the folded protein so that by measuring accurately the mass of the protein with cofactors bound, the identity of the cofactor can be deduced. This has recently provided unprecedented insight into iron-sulfur cluster conversion and degradation in other systems, and is an extremely promising, novel methodology to elucidate the steps of de novo cluster assembly.Overall, our research has important implications for our basic comprehension of these cellular processes in bacteria, which is to a large extent conserved in humans, with potential longer term medical benefits.