Figure 6.

Effect of Zn²⁺ on IscU_wt properties. (A) BLI profile for the binding between immobilized IscS and different forms of IscU (as purified, Zn_free, Zn_saturated). The concentration of IscU used in this experiment was 1 µM (below the Kd value) thus avoiding artifacts due to the saturation of IscS immobilized. (B) Kinetics of enzymatic cluster formation on IscU under strict anaerobic conditions using IscU (50 µM), IscS (1 µM), 250 µM cysteine, 3 mM DTT, and 25 µM Fe²⁺. IscU_wt was used as purified (dark green), as pretreated with EDTA (yellow), and as pretreated by EDTA to which Zn²⁺ was added (1:5; light green). (C) The same as in B but comparing the kinetics recorded using 25 µM Fe²⁺ with 250 µM Fe²⁺ and adding a 1:5 Zn²⁺ excess. (D) Effect of Zn²⁺ on the NMR HSQC spectrum of IscU_wt. From top to bottom: spectra of EDTA pretreated IscU_wt, titrated with 1:1, 5:1, and 10:1 Zn²⁺: protein molar equivalents. The spectra were recorded at 25°C and 600 MHz (Varian INOVA) in 10 mM in Tris–HCl buffer at pH 8.0.