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.