In Vitro Studies of Cellular Iron-Sulfur Cluster Biosynthesis, Trafficking, and Transport

Iron-sulfur clusters are metal cofactors that comprise the largest class of metalloproteins and are utilized for a wide variety of functions ranging from electron transport to DNA repair. These clusters and their respective cluster-binding proteins are highly conserved and are produced in the mitochondria via an evolutionarily conserved process for export to the cytosol and delivery to other organelles, including the nucleus. Disruption of the biosynthetic pathway results in a number of disease conditions that reflect the essential requirements of cluster function and trafficking within the cell. In vivo studies are limited in their ability to examine the detailed molecular mechanisms of protein-protein interactions, since they often focus on the downstream effects of protein depletion or mutation. As such, in vitro analyses are essential for defining the roles of specific Fe-S proteins in trafficking events and supporting in vivo analyses of disease conditions arising from aberrant Fe-S assembly and trafficking. In this chapter, we describe a variety of methods for the analysis of structure-function relationships in holo Fe-S cluster proteins, as well as monitoring the kinetics and molecular mechanisms of Fe-S cluster transfer.