Iron−Sulfur Cluster Biosynthesis: Characterization of Iron Nucleation Sites for Assembly of the [2Fe−2S]2+ Cluster Core in IscU Proteins

ISU (eukaryotes) and IscU (prokaryotes) are a homologous family of proteins that appear to provide a platform for assembly of [2Fe−2S] centers prior to delivery to a target apoprotein. The intermediate [2Fe−2S] IscU-bound cluster is formed by delivery of iron and sulfur to the apo-IscU, with the latter delivered through an IscS-mediated reaction. The identity of the iron donor is not yet established. In this report we characterize iron-binding sites on IscU that appear to nucleate [2Fe−2S] cluster assembly. This iron-bound form of IscU is shown to be viable for subsequent IscS-mediated assembly of holo-IscU. Following on recent reports, we demonstrate the persulfide form of IscU to be a dead-end complex that is incapable of forming holoprotein after addition of ferrous or ferric ion. The latter observation reflects the low binding affinity of persulfido IscU for iron ion.