Unraveling the mechanism of [4Fe-4S] cluster assembly on the N-terminal cluster binding site of NUBP1

[4Fe-4S]²⁺ cluster assembly in human cytosol requires both a [2Fe-2S] cluster chaperone being able to donate two [2Fe-2S]²⁺ clusters and an electron donor providing two electrons to reductively couple the two [2Fe-2S]²⁺ clusters into a [4Fe-4S]²⁺ cluster. The mechanism through which the cytosolic [4Fe-4S]²⁺ cluster assembly works is still not defined. Here, we show that a hetero-tetrameric complex formed by two molecules of cluster-reduced [2Fe-2S]⁺ ₂ -anamorsin and one molecule of dimeric cluster-oxidized [2Fe-2S]²⁺ ₂ -GLRX3₂ orchestrates the assembly of a [4Fe-4S]²⁺ cluster on the N-terminal cluster binding site of the cytosolic protein NUBP1. We demonstrate that the hetero-tetrameric complex is able to synergically provide two [2Fe-2S]²⁺ clusters from GLRX3 and two electrons from anamorsin for the assembly of the [4Fe-4S]²⁺ cluster on the N-terminal cluster binding site of NUBP1. We also showed that only one of the two [2Fe-2S] clusters bound to anamorsin, that is, that bound to the CX₈ CX₂ CXC motif, provides the electrons required to form the [4Fe-4S]²⁺ cluster. Our study contributes to the molecular understanding of the mechanism of [4Fe-4S] protein biogenesis in the cytosol.