Methods to Unravel the Roles of ATPases in Fe-S Cluster Biosynthesis

Complex biosynthetic pathways are required for the assembly and insertion of iron-sulfur (Fe-S) cluster cofactors. Each of the four cluster biogenesis systems that have been discovered requires at least one ATPase. Generally, the function of nucleotide hydrolysis in Fe-S cluster biogenesis is understudied. For example, the cytosolic Fe-S cluster assembly (CIA) pathway is proposed to begin with a scaffold, which assembles nascent Fe-S clusters destined for cytosolic and nuclear enzymes. This scaffold, comprised of Nbp35 and Cfd1 in yeast, possesses an ATPase site that is necessary for CIA function, but the role of nucleotide hydrolysis is poorly understood. Herein, we describe the in vitro methods that have been developed to uncover how the ATPase site of the scaffold regulates interaction with one of its partner proteins, Dre2. We describe a qualitative affinity copurification assay and a quantitative assay for evaluating the dissociation constant for the scaffold-partner protein complex. Finally, we describe kinetic methods to measure the kcat and KM values for ATP hydrolysis by the scaffold-partner protein complex and the execution of the ATPase assays in an anaerobic environment. These methods could be applied to study other ATPases to advance our mechanistic understanding of nucleotide hydrolases involved in metallocluster biogenesis.