Understanding the molecular mechanism of iron sulfur cluster biogenesis

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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.