Metal ions are essential to life as they augment amino acid protein chemistry and thereby catalyze many difficult biological reactions. As “free” metal ions are toxic and indiscriminately reactive, critical protein systems have evolved to sequester, chaperone, and regulate metal ion concentrations. Defects in these systems lead to metal ion metabolic disease and result in cellular, tissue, and systemic pathology. The iron-sulfur cluster assembly pathway contains a conserved set of metallochaperone proteins that recognize and insert Fe-S clusters into apo metalloproteins. We determined the first crystal structure for the NFS1-ISD11-ACP cysteine desulfurase, which is a central enzyme in this pathway that is also implicated in providing sulfur for molybdenum cofactor biosynthesis and tRNA modifications. Interestingly, three distinct interchangeable α2β22 architectures are now known for the cysteine desulfurase complex that have superimposable protomers but distinct protein-protein interactions. In this proposal, we aim to investigate the functional relationship between these architectures, elucidate mechanistic details for sulfur transfer to different acceptor proteins and for Fe-S cluster assembly, and provide new insight into the regulatory control mechanisms for human Fe-S cluster biosynthesis. This fundamental research will establish a framework for emerging genetic results and discoveries and provide a basis for understanding defects in iron-sulfur cluster metabolism relevant to human health and disease.