Application Notes

3 All titrations reached saturation and sigmoidal curves could be fitted (Fig. 2b). The results demonstrate a preferential interaction of Isu1 with reduced Yah1, thus facilitating electron transfer during de novo Fe/S cluster synthesis. We further used anaerobic MicroScale Thermophoresis to investigate the potential interaction between Yah1 and Nfs1, since the bacterial counterparts were reported to form a complex 16 . Reduced Yah1 showed only a weak interaction with Nfs1-Isd11, in the K d range of the complex of oxidized Yah1 and Isu1 (Fig. 2a, c). Surprisingly, a tenfold higher affinity was observed for oxidized Yah1 and Nfs1- Isd11 (Fig. 2a, c), suggesting a dynamic rearrangement of Yah1 within the ISC biosynthetic complex after electron transfer. Notably, the binding between reduced Yah1 and apo-Isu1 was still 10-fold stronger than that between oxidized Yah1 and Nfs1-Isd11, emphasizing the physiological relevance of reduced Yah1−apo- Isu1 interaction for initiating Fe/S cluster synthesis. Discussion MicroScale Thermophoresis allowed anaerobic equilibrium titrations, which has never been possible before. This method identified a strong interaction (K d = 25 nM) between reduced Yah1 and apo-Isu1. A 100-fold weaker affinity was observed for oxidized Yah1 and apo-Isu1 (Δ(ΔG) Ox/Red = 2.9 kcal/mol). The high preference of apo-Isu1 for the reduced form of Yah1 provides strong evidence for the physiologically relevant encounter of the two proteins. Additionally, anaerobic thermophoresis revealed a higher affinity of oxidized Yah1 to Nfs1-Isd11 than to Isu1, suggesting that upon oxidation of Yah1 it loosens the interaction with Isu1 and undergoes tighter binding with Nfs1-Isd11. These results suggest a so far undescribed dynamic rearrangement of the five-membered ISC complex during Fe/S cluster formation. Methods Affinity measurements using anaerobic MicroScale Thermophoresis (MST). MST was performed on a Monolith NT.115 (NanoTemper Technologies GmbH, Munich, Germany) at 32 °C or 28 °C (LED power (red channel) was set between 60 % and 100 % and MST power to 80 %) 33 . All samples and dilution series were prepared anaerobically in a COY anaerobic chamber (95 % N 2 ; 5 % H 2 ). Samples were filled in the capillaries and wax-sealed prior to measurement. Isu1 or Nfs1-Isd11 (20 µM each) were labeled with the dye NT647 according to the supplier (NanoTemper Technologies). 200 nM Isu1 or Nfs1-Isd11 were titrated with unlabeled Yah1, FDX2, FDX1 or Yfh1 in 35 mM KP i pH 7.4, 150 mM NaCl. At least six independent MST experiments were recorded at 680 nm and processed by NanoTemper Analysis 1.2.009 and Origin8. References For the complete set of data and detailed information please refer to Webert et al. 1 . 1 Webert, H. et al. Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin. Nat Commun 5, 5013, doi:10.1038/ncomms6013 (2014). 2 White, M. F. & Dillingham, M. S. Iron-sulphur clusters in nucleic acid processing enzymes. Current opinion in structural biology, doi:10.1016/j.sbi.2011.11.004 (2011). 3 Crack, J. C., Green, J., Thomson, A. J. & Le Brun, N. E. Iron-sulfur cluster sensor-regulators. Current opinion in chemical biology 16, 35-44, doi:10.1016/j.cbpa.2012.02.009 (2012). 4 Beinert, H. Iron-sulfur proteins: ancient structures, still full of surprises. J. Bioinorg. Chem. 5, 2-15. (2000). 5 Rouault, T. A. Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease. Disease models & mechanisms 5, 155-164, doi:10.1242/dmm.009019 (2012). 6 Sheftel, A., Stehling, O. & Lill, R. Iron-sulfur proteins in health and disease. Trends Endocrinol Metab 21, 302-314. (2010).

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