Exploring the impact of molecular crowding and iron on IscS enzyme kinetics: bridging in vitro and ex vivo perspectives

IscS/Nfs1 desulfurase plays a pivotal role in iron-sulfur (Fe-S) cluster biogenesis, a process essential for cellular function across all domains of life. In prokaryotes and eukaryotes, the protein CyaY/Frataxin exhibits opposing effects on IscS/Nfs1, acting as an inhibitor or activator, respectively. This functional discrepancy has been primarily observed under in vitro conditions, where simplified systems may not fully replicate the cellular environment, potentially leading to misleading conclusions. The research presented in this thesis aims to bridge this gap by investigating the function and regulation of the IscS desulfurase in a more physiologically relevant context. The study focuses on understanding the enzyme's behavior in crowded cellular environments, mimicking real-cell conditions using bacterial lysates and agarose as crowding agents. By analyzing enzyme kinetics under these conditions, the aim is to uncover how molecular crowding influences enzyme activity, substrate binding, and the regulatory role of iron in Fe-S cluster biogenesis. The findings highlight the impact of cellular complexity on enzyme function, providing insights into the molecular mechanisms that govern IscS activity. These insights are crucial for advancing our understanding of Fe-S cluster biogenesis and could inform the development of therapeutic strategies targeting related pathologies, such as Friedreich's ataxia, where frataxin deficiency leads to impaired Fe-S cluster formation and subsequent cellular dysfunction. The thesis demonstrates that the crowded cellular environment significantly affects IscS activity, with implications for both basic biological research and clinical applications. By elucidating the enzyme's regulation under near-physiological conditions, this work contributes to a deeper understanding of cellular metabolism and the potential for therapeutic intervention in diseases involving Fe-S cluster assembly defects.