CIA secrets revealed: mechanistic insights into the early steps in the cytosolic iron sulfur cluster assembly pathway

Seminar | May 15 | 11 a.m.-12:30 p.m. | 100 Lewis Hall

 Deborah Perlstein, Department of Chemistry, Boston University

 College of Chemistry

The cytosolic iron sulfur cluster assembly (CIA) scaffold is comprised of two homologous ATPases, called Nbp35 and Cfd1 in yeast. These proteins cooperate to build new [Fe4S4]-clusters destined for cytosolic and nuclear enzymes. Genetic studies have revealed that the ATPase sites are required for cluster biogenesis in vivo, but in vitro studies have failed to reveal why. Since several distantly related NTPases serve as central regulatory switches in which nucleotide binding and hydrolysis regulate dynamic interactions with small molecule effectors and/or protein interaction partners, we investigated how FeS cluster and other CIA factors impact the scaffold’s ATP affinity and hydrolysis activity. We found that the holo-scaffold, which has chemically reconstituted FeS clusters, binds ATP with diminished affinity but the kinetics of ATP hydrolysis remain largely unchanged. In contrast, we found that Dre2, a reductase proposed to provide reducing equivalents to the scaffold, and Nar1, a CIA factor of unknown function, both stimulate the scaffold’s ATP hydrolysis activity. Dre2 binds Nbp35-Cfd1 complex with low micromolar affinity and increases the kcat for ATP hydrolysis ~20-fold. Based on our in vitro and in vivo studies we propose a model for the first half of the scaffold reaction cycle where ATP binds first, initiating a conformational change in the scaffold. This ATP-bound state is competent to bind Dre2 and complete cluster biogenesis. Current work is focused on defining how Nar1 affects the ATPase reaction cycle in an effort to understand the fate of the scaffold’s newly built FeS cluster.