Cluster exchange reactivity of [2Fe‐2S]‐bridged heterodimeric BOLA1‐GLRX5

Mitochondrial BOLA1 is known to form a [2Fe‐2S] cluster‐bridged heterodimeric complex with mitochondrial monothiol glutaredoxin GLRX5; however, the function of this heterodimeric complex is unclear. Some reports suggest redundant roles for BOLA1 and a related protein, BOLA3, with both involved in the maturation of [4Fe‐4S] clusters in a subset of mitochondrial proteins. However, a later report on the structure of BOLA1‐GLRX5 heterodimeric complex demonstrated a buried cluster environment and predicted a redox role instead of the cluster trafficking role suggested for the BOLA3‐GLRX5 heterodimeric complex. Herein, we describe a detailed kinetic study of relative cluster exchange reactivity involving heterodimeric complex of BOLA1 with GLRX5. By the use of CD spectroscopy, it is demonstrated that [2Fe‐2S]‐bridged BOLA1‐GLRX5 can be readily formed by cluster uptake from donors such as ISCU or [2Fe‐2S](GS)4 complex, but not from ISCA1 or ISCA2. Rapid holo‐formation following delivery from [2Fe‐2S](GS)4 supports possible physiological relevance in the cellular labile iron pool. Holo [2Fe‐2S] BOLA1‐GLRX5 heterodimeric complex is incapable of donating cluster to apo protein acceptors, providing experimental support for a nontrafficking role. Finally, we report the formation and reactivity of the holo [2Fe‐2S]‐bridged BOLA1 homodimer (lacking a partner GLRX). While the holo‐heterodimer is thermodynamically more stable, by contrast the holo BOLA1 homodimer does demonstrate facile cluster exchange reactivity. BOLA1‐GLRX5 apo heterodimer accepts cluster from physiological donors such as [2Fe‐2S](GS)4 with the rapid formation of [2Fe‐2S]‐bridged BOLA1‐GLRX5. In contrast to cluster‐bridged BOLA3‐GLRX5 heterodimer, cluster‐bridged BOLA1‐GLRX5 heterodimer is incapable of downstream cluster trafficking. Heterodimers of human BOLA1 serve a different cellular role compared with BOLA3.