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. Author manuscript; available in PMC: 2020 Jun 1.
Published in final edited form as: J Am Chem Soc. 2020 Mar 17;142(13):6018–6029. doi: 10.1021/jacs.9b11454

Figure 3.

Figure 3.

Zn2+ inhibits Fe–S assembly activity and is removed by IscS-mediated cysteine turnover. Parallel cluster formation reactions using (A) Zn–IscU or (B) apo–IscU were monitored by CD spectroscopy. Experimental conditions: 100 μM IscU, 3 μM IscS, 200 μM L-cysteine, 100 μM GSH, and 500 μM ferrous acetate. (C) Deconvoluted zero charge spectra for removal of Zn2+ from Zn–IscU (10 μM) monitored by native MS after incubations with L-cysteine (1 mM), D,L-DTT (1 mM), and GSH (200 μM). Similar experiments were performed after incubation of Zn–IscU (40 μM) with IscS (40 μM), ferrous acetate (800 μM), IscS (40 μM) plus Fe2+ (200 μM), and IscS (40 μM) plus L-cysteine (200 μM). All reactions were incubated for 30 min at room temperature except for the combination of Zn–IscU with IscS and L-cysteine, which was reacted for 5 min. All experiments were repeated at least twice, and similar results were obtained.