A pathway for assembling [4Fe‐4S] 2+ clusters in mitochondrial iron–sulfur protein biogenesis

During its late steps, the mitochondrial iron–sulfur cluster (ISC) assembly machinery leads to the formation of [4Fe‐4S] clusters. In vivo studies revealed that several proteins are implicated in the biosynthesis and trafficking of [4Fe‐4S] clusters in mitochondria. However, they do not provide a clear picture into how these proteins cooperate. Here, we showed that three late‐acting components of the mitochondrial ISC assembly machinery (GLRX5, BOLA3, and NFU1) are part of a ISC assembly pathway leading to the synthesis of a [4Fe‐4S] 2+ cluster on NFU1. We showed that the [2Fe‐2S] 2+ GLRX5‐BOLA3 complex transfers its cluster to monomeric apo NFU1 to form, in the presence of a reductant, a [4Fe‐4S] 2+ cluster bound to dimeric NFU1. The cluster formation on NFU1 does not occur with [2Fe‐2S] 2+ GLRX5, and thus, the [4Fe‐4S] cluster assembly pathway is activated only in the presence of BOLA3. These results define NFU1 as an ‘assembler’ of [4Fe‐4S] clusters, that is, a protein able of converting two [2Fe‐2S] 2+ clusters into a [4Fe‐4S] 2+ cluster. Finally, we found that the [4Fe‐4S] 2+ cluster bound to NFU1 has a coordination site which is easily accessible to sulfur‐containing ligands, as is typically observed in metallochaperones. This finding supports a role for NFU1 in promoting rapid and controlled cluster‐exchange reaction.