Fig. 5: The N-terminal Tyr35 of ISCU2 is not required for persulfide reduction, yet essential for [2Fe-2S] cluster synthesis and ISCU2 dimerization.
From: N-terminal tyrosine of ISCU2 triggers [2Fe-2S] cluster synthesis by ISCU2 dimerization
a Reduction of ISCU2-Y35A-bound persulfide by FDX2 assayed as described in Fig. 3c (see also Supplementary Fig. 7a). Reactions without (NIA)2 or cysteine served as controls (lanes 1 + 2 and 3 + 4, respectively). The re-appearance of fourfold MPB-labeled ISCU2 in lanes 6 and 8 indicates efficient persulfide reduction by FDX2. The experiment was repeated twice with similar outcome. b Persulfide transfer from NFS1 onto wild-type ISCU2 as well as ISCU2-Y35D or ISCU2-Y35K mutant proteins. Reactions without (NIA)2 plus and minus MPB served as controls. The experiment was repeated at least three times with similar outcome. c Enzymatic reconstitution (cf. Fig. 3d) of wild-type ISCU2 or the ISCU2 mutant proteins Y35D, Y35K, and the mixture of Y35D and Y35K as indicated. d Anaerobic analytical size-exclusion chromatography (aSEC) of enzymatically reconstituted ISCU2 and variants (color coding as in b). The majority of ISCU2 and ca. 50% of the Y35D + Y35K mixture form dimers that carry a [2Fe-2S] cluster as indicated by absorption at 320 nm and 420 nm and CD spectrum (Supplementary Fig. 9a). In contrast, ISCU2-Y35D and ISCU2-Y35K eluted as monomers like apo-ISCU2, and showed no absorbance at 320 nm or 420 nm indicating that no Fe/S cluster was formed. Source Data are provided as a Source Data file.
