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2 Results Reduced mitochondrial ferredoxin Yah1 interacts with Isu1 The physiological interaction partner of Yah1 within the mitochondrial ISC assembly machinery has remained elusive. Yah1 is predicted to interact in its reduced state with Isu1 to productively deliver electrons for Fe/S cluster synthesis. Because determining a K d anaerobically via equilibrium titration has only been possible since MicroScale Thermophoresis was launched, the influence of the Yah1 redox state for Isu1 interaction was addressed by this novel technique the first time 15 . Increasing amounts of reduced or oxidized Yah1 were mixed with fluorescently labelled Isu1 under anaerobic conditions and the thermophoretic behaviour was followed using wax-sealed capillaries on a Monolith NT.115. Isu1 strongly interacted with reduced Yah1 (K d = 25 nM), yet 100-fold weaker with oxidized Yah1 (Fig. 2a). This result made it possible to determine the interaction interface on Yah1 via NMR (Fig. 1). a b 0 500 1000 1500 2000 2500 3000 K d [nM] c K d = 25.5 nM K d = 2.4 µM K d = 240 nM K d = 2.5 µM Isu1+Yah1-Red Isu1+Yah1-Ox Nfs1-Isd11+Yah1-Red Nfs1-Isd11+Yah1-Ox Figure 2: Quantitative determination of the interaction between the scaffold protein Isu1 or the cysteine desulfurase complex Nfs1-Isd11 with Yah1. (a) Bar chart showing K d . Isu1 preferentially interacts with reduced Yah1 while Nfs1-Isd11 complex better interacts with oxidized Yah1. Yah1 was used as isolated (Yah1(ox)) or reduced with dithionite (Yah1(red)), and mixed at increasing concentrations with 200 nM fluorescently labeled Isu1 or Nfs1-Isd11. MicroScale Thermophoresis experiments were performed, and dissociation constants (K d ) were determined (Error bars indicate the SD (n=6). (b) Isu1 or Nfs-Isd11 was labelled with the fluorescent dye NT 647 (NanoTemper Technologies, Munich Germany). All steps were performed under anaerobic conditions. Yah1 was reduced with sodium dithionite at a 5-fold molar excess. After incubation for 25 min dithionite was removed using Zeba Spin desalting columns (Thermo Scientific). Fluorescently labelled Isu1 or Nfs1/Isd11 (final concentration of 200 nM) was added to the indicated concentrations of Yah1. The MST binding reactions were measured using Monolith NT.115 at 32 °C or 28 °C for Isu1-Yah1 and Nfs1- Isd11-Yah1, respectively. (LED power: 100 %, MST power: 80 %). The K d values were estimated from at least six independent experiments using NanoTemper Analysis 1.5.37 and Origin 8G software.

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