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Iron-sulfur (Fe/S) clusters are ancient protein cofactors and ubiquitously present in all kingdoms of life. Fe/S-proteins coordinate essential functions within the cell, e.g. cellular respiration, DNA-synthesis and protein translation. Despite the relatively simple chemical structure and composition of Fe/S-clusters, the cell requires a complex system for their synthesis and incorporation into apo-proteins. Deficiencies in Fe/S-protein biogenesis are often linked with severe metabolic defects and serious diseases such as Friedreich‘s ataxia or sideroplastic anemia. A better understanding of the biogenesis of Fe/S-proteins may help to find new therapies for such life-threatening diseases.
In eukaryotes, the synthesis of Fe/S-clusters starts inside mitochondria (or related organelles such as mitosomes). These organelles contain the so-called „iron-sulfur-cluster“ (ISC) assembly machinery. The early part of the ISC-machinery accomplishes the synthesis of a [2Fe-2S]-cluster on the scaffold protein Isu1 which requires five additional factors. The desulfurase complex Nfs1/Isd11 delivers the sulfur while the frataxin Yfh1 serves as an iron-donor and/or sulfur-carrier. The so provided iron and sulfur will finally be assembled to an Fe/S-cluster. The assembly process depends on an electron supply by the ferredoxin Yah1 and its reductase Arh1. In this study, additional mechanistic and structural insights into the early ISC-machinery were gained at a molecular level. First, it was shown how the two proteins interact, based on the atomic structures of Yah1, Isu1 and the Yah1-Isu1 complex solved by NMR-spectroscopy. This revealed a possible mechanism for the electron transfer from the reduced Fe/S-cluster of Yah1 to Isu1. Therefore, the structural changes of Yah1 upon reduction play a major role while the essential His51 may have an electron-bridging function. Second, biochemical studies of the interactions of all early ISC factors by Thermophoresis contributed to a time-resolved model of the mechanism of the initial Fe/S-cluster biogenesis. All primary ISC factors interact with each other and form a biologically competent complex, which is dependent on both the redox-state of the components and the presence of Fe(II) and cysteine. Finally, the overall structure of the early ISC-machinery was determined by SAXS (small angle X-ray scattering). This allowed the early ISC complex to be defined as a dodecamer having the following stoichiometry: 2(Nfs1+2Isd11+Yfh1+Isu1+Yah1). These structural data, despite the relatively low resolution, allowed new insightful conclusions about the molecular mechanism of Fe/S-cluster biogenesis. The Yah1-Isu1 complex is located in close proximity to the essential persulfide-bearing cysteine residue of Nfs1, and thus enables a persulfide-transfer from Nfs1 to Isu1. Then the persulfide can be reduced by Yah1. The previously proposed function of the frataxin Yfh1 as an iron-donor was further substantiated due to its localization within the ISC-complex and in particular the position of its conserved histidine 23. For the first time the location of Isd11 on Nfs1 could be shown. Isd11 binds far away from the active site of the ISC-complex, but is located in close proximity to the active site of Nfs1 and its cysteine binding pocket. This leads to the conclusion that Isd11 may regulate the activity of Nfs1. Thus, these data offer an explanation for the origin and the transfer mechanism of both the iron and the sulfur on a molecular basis and suggest a possible location of Isd11 on Nfs1. Taken together, these results constitute an important step in the direction of the molecular understanding of the Fe/S cluster biogenesis and therefore also form the basis for further investigations.