Alternative [ 2Fe ‐ 2S ] donor assemblies in the cytosol: GLRX3 and GLRX

Iron–sulfur (Fe‐S) cluster assembly in the cytosol relies on coordinated transfer of [2Fe‐2S] precursors to scaffold proteins of the cytosolic iron–sulfur assembly (CIA) machinery. Human glutaredoxin 3 (GLRX3) functions as a central [2Fe‐2S] cluster donor and can form a heterotrimeric complex with the BolA‐like protein BOLA2, whose functional role in cluster trafficking remains unclear. Here, we directly compare the cluster donor properties of the GLRX3 homodimer with those of the GLRX3‐BOLA2 heterotrimer in the maturation of the CIA scaffold nucleotide‐binding protein 1 (NUBP1). Using a combination of UV–Vis spectroscopy, acid‐labile iron and sulfide quantification, circular dichroism kinetics, and paramagnetic NMR, we show that both complexes support assembly of a canonical [4Fe‐4S] cluster on the N‐terminal site of NUBP1. The structural and spectroscopic features of the assembled cluster are indistinguishable between the two donor systems, and comparable levels of cluster incorporation are observed. Kinetic analysis reveals that cluster transfer proceeds with similar apparent rate constants for GLRX3 and GLRX3‐BOLA2 under glutathione‐supported conditions. The presence of the physiological electron donor anamorsin accelerates the GLRX3‐mediated transfer but does not significantly affect the kinetics of the GLRX3‐BOLA2 system, suggesting a shift in the rate‐limiting step upon BOLA2 association. Together, these results show that GLRX3‐BOLA2 is fully competent for [2Fe‐2S] cluster delivery to NUBP1 and supports the formation of the same final [4Fe‐4S] cluster observed upon GLRX3‐mediated transfer. Rather than enhancing transfer efficiency, BOLA2 association appears to play a nonkinetic role, potentially related to cluster stabilization by modulating the electron transfer process under specific cellular conditions.