Coupling Nucleotide Binding and Hydrolysis to Iron-Sulfur Cluster Acquisition and Transfer Revealed through Genetic Dissection of the Nbp35 ATPase Site.

Abstract

The cytosolic iron-sulfur cluster assembly (CIA) scaffold, comprising Nbp35 and Cfd1 in yeast, assembles iron-sulfur (FeS) clusters destined for cytosolic and nuclear enzymes. ATP hydrolysis by the CIA scaffold plays an essential but poorly understood role in cluster biogenesis. Here we find that mutation of conserved residues in the four motifs comprising the ATPase site of Nbp35 diminished the scaffold's ability to both assemble and transfer its FeS cluster in vivo. The mutants fall into four phenotypic classes that can be understood by how each set of mutations affects ATP binding and hydrolysis. In vitro studies additionally revealed that occupancy of the bridging FeS cluster binding site decreases the scaffold's affinity for the nucleotide. On the basis of our findings, we propose that nucleotide binding and hydrolysis by the CIA scaffold drive a series of protein conformational changes that regulate association with other proteins in the pathway and with its newly formed FeS cluster. Our results provide insight into how the ATPase and cluster scaffolding activities are allosterically integrated.