PROJECT SUMMARY Iron-sulfur (Fe-S) clusters are ancient cofactors composed of multiple iron and sulfur atoms. They are fundamental to numerous biological processes in all domains of life. Owing to the rich, tunable redox reactivity and selectivity of the cluster, Fe-S proteins play multifaced roles in redox control under both physiological and stress conditions. The roles of Fe-S proteins in redox control are vital for the maintenance of normal cellular functions and cell survival, and thus they are tightly linked to health and disease such as cancer and diabetes in human and bacterial infection. This contrasts vividly with the lack of functional, structural and mechanistic understanding of many Fe-S proteins in the cellular control of redox homeostasis, particularly in the three core aspects that are addressed in this MIRA proposal: i) redox sensing and transcriptional regulation by Fe-S proteins; ii) assembly, transfer and repair of Fe-S clusters; and iii) crosstalk between Fe-S proteins and the low-molecular- weight (LMW) thiols in redox hemostasis. Several Fe-S proteins-mediated mechanisms for redox control in mycobacteria will be used as examples to elaborate our research goals and approaches in this proposal, including i) redox sensing and transcriptional regulation by a unique family of Fe-S cluster-bound transcription factors in the WhiB-like family; ii) assembly, transfer and repair of Fe-S clusters by the SUF system; and iii) mycothiol in Fe-S cluster homeostasis. The proposed research program is built on the unique combination of skills and rich research experience in my research team that are crucial for characterizing the oxygen-sensitive metal-binding proteins. We recently determined the first and long-desired Fe-S cluster-bound structure of the monomeric transcription factor WhilB1 from Mycobacterium tuberculosis and established a new mechanism of bacterial transcriptional regulation mediated by this protein. By combining structural, spectroscopic and biochemical approaches in vitro with molecular biology in vivo, we are poised to determine: i) the structural basis of redox reactivity and ligand selectivity in Fe-S clusters; ii) the mechanism by which the redox state and integrity of the Fe-S cluster allows these proteins to sense redox state and regulate transcription; iii) the structural biochemistry of Fe-S cluster biosynthesis and regulation; and iv) the role of non-proteinaceous thiols in modulating Fe-S cluster-mediated redox control. Altogether, the proposed research program will establish a new line of ground-breaking research in an understudied aspect of redox homeostasis. The strategies developed from the proposed program will be instrumental for studies on the newly discovered Fe-S cluster system, such as those in the DNA repair and RNA metabolism in response to oxidative stress. Because of their critical roles in redox control, the study of the novel mechanisms of these Fe-S proteins will not only shed light on the fund...