Fig. 3: No crucial role of Trp97 of NFS1 in early steps of core ISC complex function.

a Affinities of NFS1 or indicated NFS1-W97 mutant proteins (as part of purified (NIA)₂) for ISCU2 were estimated by microscale thermophoresis. The numbers within the bars represent K_d values (error bars represent SD of the fit using three independent biological replicates, Supplementary Fig. 6c). b Determination of DTT-mediated sulfide (S^2-) production by the cysteine desulfurase NFS1 or indicated mutant proteins in the presence of ISCU2 without and with FXN (n = 3 biological replicates, individual data points are shown, error bars show SD). c Persulfide transfer from NFS1 or the indicated NFS1-W97 mutant proteins (with added FXN) onto ISCU2 was assayed by Cys alkylation with maleimide-polyethylene-glycol₁₁-biotin (MPB) as described in Supplementary Fig. 7a. Samples were analyzed by SDS-PAGE. As controls, the running behavior of ISCU2 minus and plus MPB alkylation and Cys is shown in the first two lanes. d Representative data for enzymatic Fe/S cluster reconstitution on ISCU2 with NFS1 or indicated NFS1-W97 mutant proteins. As a control, FXN was omitted for the wild-type reaction (no FXN). Dashed lines indicate the period used for initial rate determination (Supplementary Fig. 7b). (n = 3 biological replicates) e Gal-NFS1 yeast cells were transformed with vectors (low copy, native promoter) encoding either wild-type Nfs1 (WT), no protein, or Nfs1-W138A mutant protein (yeast Nfs1-W138 corresponds to human NFS1-W97). Cells were grown on glucose for 24 h to deplete endogenous Nfs1, and were subsequently spotted onto SC agarose plates containing galactose (Gal) to promote expression of chromosomal NFS1 or glucose (Glc) or glycerol (Gly) to repress NFS1 expression. A fivefold serial dilution of cells was grown for 3 days at 30 °C. Source Data are provided as a Source Data file.