Abstract
Human cytosolic monothiol glutaredoxin-3 (GLRX3) is a protein essential for the maturation of cytosolic [4Fe-4S] proteins. We show here that dimeric cluster-bridged GLRX3 transfers its [2Fe-2S](2+) clusters to the human P-loop NTPase NUBP1, an essential early component of the cytosolic iron-sulfur assembly (CIA) machinery. Specifically, we observed that [2Fe-2S](2+) clusters are transferred from GLRX3 to monomeric apo NUBP1 and reductively coupled to form [4Fe-4S](2+) clusters on both N-terminal CX(13)CX(2)CX(5)C and C-terminal CPXC motifs of NUBP1 in the presence of glutathione that acts as a reductant. In this process, cluster binding to the C-terminal motif of NUBP1 promotes protein dimerization, while cluster binding to the N-terminal motif does not affect the quaternary structure of NUBP1. The cluster transfer/assembly process is not complete on both N- and C-terminal motifs and indeed requires a reductant stronger than GSH to increase its efficiency. We also showed that the [4Fe-4S](2+) cluster formed at the N-terminal motif of NUBP1 is tightly bound, while the [4Fe-4S](2+) cluster bound at the C-terminal motif is labile. Our findings provide the first evidence for GLRX3 acting as a [2Fe-2S] cluster chaperone in the early stage of the CIA machinery.