Iron-sulfur clusters are one of the most primitive protein cofactors, present in all three kingdoms of life and performing diverse functions such as electron transfer, DNA repair and replication, gene regulation and catalysis. The biosynthesis of such iron-sulfur clusters involves highly regulated cluster assembly pathways, which include ISC (Iron-Sulfur Cluster), SUF (Sulfur Utilization Factor), CIA (Cytosolic Iron-sulfur Assembly) and NIF (Nitrogen Fixation) pathways. The ISC pathway is the most important cluster assembly pathway in mammals as it is responsible for cluster maturation of mitochondrial as well as cytosolic proteins. The ISC pathway involves de novo synthesis of a [2Fe-2S] cluster on scaffold protein Isu, which is then trafficked and delivered to apo protein targets. It has been well established that homodimeric monothiol glutaredoxins Grx5 (mitochondrial) and Grx3 (cytosolic) play a crucial role as intermediary cluster carriers by binding a [2Fe-2S] at the dimer interface with glutathione also coordinating to Fe center. Monothiol glutaredoxins can also bind [2Fe-2S] cluster in the form of a heterodimeric complex with another partner protein belonging to the BolA family. Though exact role of the BolA proteins is not known, previous studies have proposed that the [2Fe-2S]-bound bridged complexes of glutaredoxin Grx5 with BolA1 and BolA3 may have distinct and essential physiological roles in humans. Genetic mutations in BolA3 cause the fatal disease Multiple Mitochondrial Dysfunctions Syndrome 2 (MMDS2), though the molecular reason for the disease condition remains unknown.
The focus of this work is to understand the mechanistic details of [2Fe-2S] cluster trafficking by monothiol glutaredoxin along with its partner BolA protein. The seemingly simple process of cluster exchange involving such entities potentially involves multiple Fe-cysteinyl bond dissociation and formation events, accompanied by simultaneous exchange of glutathione molecules. Herein, I have adopted systematic approach to dissect the steps of the cluster exchange reaction, involving homodimeric glutaredoxins, and also investigated the cluster lability and exchange reactivity of heterodimeric complexes of glutaredoxins and BolA proteins. I have also been able to demonstrate that the formation of the heterodimeric complex of Grx5 and BolA3 is affected in the MMDS2 disease mutant of BolA3, thereby explaining the probable molecular basis of the disease condition. Lastly, I have been able to identify that monothiol glutaredoxins can accept cluster from the mitoNEET protein, a protein which is characterized by its unique [2Fe-2S] lability.