The Escherichia coli protein IscU serves as the scaffold for Fe-S cluster assembly and the vehicle for Fe-S cluster transfer to acceptor proteins, such as apoferredoxin. IscU populates two conformational states in solution, a structured conformation (S) that resembles the conformation of the holoprotein IscU-[2Fe-2S] and a dynamically disordered conformation (D) that does not bind metal ions. NMR spectroscopic results presented here show that the specialized Hsp70 chaperone (HscA), alone or as the HscA-ADP complex, preferentially binds to and stabilizes the D-state of IscU. IscU is released when HscA binds ATP. By contrast, the J-protein HscB binds preferentially to the S-state of IscU. Consistent with these findings, we propose a mechanism in which cluster transfer is coupled to hydrolysis of ATP bound to HscA, conversion of IscU to the D-state, and release of HscB.