Figure 5.
Ind1 binds a labile Fe–S cluster in vitro. (A) Binding of an [4Fe–4S] cluster to Ind1 in vitro as revealed by UV–Vis spectroscopy. Recombinant Ind1–strep (0.85 mg/ml, 26 μM) was reconstituted and desalted as described in Materials and methods. UV–Vis spectra were recorded in 100 mM Tris–HCl pH 9.0, 100 mM NaCl in the presence and absence of 2 mM sodium dithionite (as indicated). (B) Ind1 can efficiently transfer an Fe–S cluster to a model substrate, apo-isopropylmalate isomerase (Leu1). Reconstituted Ind1 (4 μM) was mixed with reduced apo-Leu1 (4 μM) under anaerobic conditions and Leu1 enzyme activity was measured at regular intervals (circles). As a control, activation of 4 μM apo-Leu1 with ferric ammonium citrate and Li2S at concentrations identical to those present in Ind1 was carried out (squares).