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Anaerobic MicroScale Thermophoresis reveals the Redox dependency of ferredoxin in mitochondrial Fe/S biogenesis

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1 Anaerobic MicroScale Thermophoresis Application Note NT-MO-026 Anaerobic MicroScale Thermophoresis reveals the Redox dependency of ferredoxin in mitochondrial Fe/S biogenesis Sven-Andreas Freibert 1,2 , Holger Webert 1 , Nina Schlinck 3 , Roland Lill 1,2,4 1 Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch-Straße 6, 35032 Marburg, Germany 2 LOEWE Zentrum für Synthetische Mikrobiologie SynMikro, Hans-Meerwein-Str., 35043 Marburg, Germany 3 NanoTemper Technologies GmbH, Flößergasse 4, 81369 München, Germany 4 Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Str. 10, 35043 Marburg, Germany Abstract Maturation of iron-sulphur (Fe/S) proteins involves a complex biosynthetic machinery. Here we investigate the interaction of one component, Yah1, with its interaction partners apo-Isu1 and Nfs1-Isd11. Using anaerobic MicroScale Thermophoresis (MST) we show the redox dependency of the interaction between Yah1 and Isu1 as well as between Yah1 and Nfs1-Isd11. Reduced but not oxidized Yah1 tightly interacts with apo-Isu1 indicating a dynamic interaction between Yah1−apo-Isu1. Introduction Iron-sulphur clusters (Fe/S) are one of the oldest inorganic prosthetic groups on earth. They play important roles in fundamental cellular processes such as respiration, amino acid and cofactor metabolism, DNA metabolism and repair, and the regulation of environmental responses 2-4 . The mitochondrial ISC (iron-sulphur cluster assembly) machinery is crucial for the synthesis of all Fe/S clusters (mitochondrial, cytosolic and nuclear) as well as for cellular iron regulation. Mutations in many mitochondrial ISC genes cause neurodegenerative, haematological and metabolic diseases with complex clinical and biochemical phenotypes 5,6-8 . The first step of Fe/S protein maturation in yeast crucially requires the scaffold protein Isu1, the cysteine desulfurase complex Nfs1-Isd11 9 and the [2Fe-2S] ferredoxin Yah1 (Fig. 1) 10-14 . So far, the exact nature of the interactions within the machinery has not been resolved in detail. Figure 1: NMR solution structure of the yeast ferredoxin Yah1 (PDB: 2MJE), in its reduced state and with its flexible C- terminus. The [2Fe-2S] cluster is represented as stick model. Isu1-interacting residues are in sphere-representation.

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