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)‐GLRX3(2) 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 CX(8)CX(2)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.