In vitro activation of apo-aconitase using a [4Fe-4S] cluster-loaded form of the IscU [Fe-S] cluster scaffolding protein

Genetic experiments have established that IscU is involved in maturation of Fe-S] proteins that require either 2Fe-2S] or 4Fe-4S] clusters for their biological activities. Biochemical studies have also shown that one 2Fe-2S] cluster can be assembled in vitro within each subunit of the IscU homodimer and that these clusters can be reductively coupled to form a single 4Fe-4S] cluster. In the present work, it is shown that the 4Fe-4S] cluster-loaded form of A. vinelandii IscU, but not the 2Fe-2S] cluster-loaded form, can be used for intact cluster transfer to an apo form of A. vinelandii aconitase A, a member of the monomeric dehydratase family of proteins that requires a 4Fe-4S] cluster for enzymatic activity. The rate of 4Fe-4S] cluster transfer from IscU to apo-aconitase A was not affected by the presence of the HscA/HscB co-chaperone system and MgATP. However, an altered form of a 4Fe-4S] cluster-containing IscU, having the highly conserved aspartate-39 residue substituted with alanine, is an effective inhibitor of wild-type 4Fe-4S] cluster-loaded IscU-directed activation of apo-aconitase A. In contrast, neither the clusterless form of IscU nor the 2Fe-2S] cluster-loaded form of IscU is an effective inhibitor of IscU-directed apo-aconitase A activation. These results are interpreted to indicate that the 2Fe-2S] and 4Fe-4S] cluster-loaded forms of IscU adopt different conformations that provide specificity with respect to the maturation of 2Fe-2S] and 4Fe-4S] centers in proteins.