Protein-Interaction Affinity Gradient Drives [4Fe–4S] Cluster Insertion in Human Lipoyl Synthase

[Image: see text] Human lipoyl synthase (LIAS) is an enzyme containing two [4Fe–4S] clusters (named FeS(RS) and FeS(aux)) involved in the biosynthesis of the lipoyl cofactor. The mechanism by which a [4Fe–4S] cluster is inserted into LIAS has thus far remained elusive. Here we show that NFU1 and ISCA1 of the mitochondrial iron–sulfur cluster assembly machinery, via forming a heterodimeric complex, are the key factors for the insertion of a [4Fe–4S] cluster into the FeS(RS) site of LIAS. In this process, the crucial actor is the C-domain of NFU1, which, by exploiting a protein-interaction affinity gradient increasing from ISCA1 to LIAS, drives the cluster to its final destination.