Fig. 2.

Activity assay analysis of wild-type ISCU in zinc-depleted and zinc-bound forms. (A) The methylene blue assay was used to determine the effect of desulfurase activity on the two states of ISCU using buffer supplemented with either 100 μM EDTA or 50 μM ZnCl₂. When noted, concentration of NFS1-ISD11-ACP (SDA) was at 0.5 μM, ISCU (U) at 2.5 μM, and Frataxin (F) at 40 μM. (B) The three conserved cysteine residues on ISCU were mutated to alanine (single variant C138A, double variant C69A-C95A) to determine if there was still a zinc-based inhibition of desulfurase activity. (C) In order to determine IC₅₀ values for zinc to the NFS1-ISD11-ACP-ISCU (SDAU) complex, we treated ISCU with EDTA to remove any zinc and then buffer exchanged to remove EDTA into buffer without EDTA or ZnCl₂. The methylene blue assay was then used in the presence of buffer containing a serial dilution of zinc concentrations and plotting [inhibitor] vs. response (inset shown on a log scale) to determine an IC₅₀ of 0.90 ± 0.06 μM for ZnCl₂ to SDAU. Error bars denote standard deviations determined from experimental replicates of n = 3.