Elucidating the mechanisms of [4Fe-4S] cluster insertions into the cytosolic iron-sulfur protein assembly component Nar1

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Dedicated cofactor trafficking and insertion pathways are an important aspect in a multitude of biosynthetic and metabolic reactions. The cell has various mechanisms for specifically determining which cofactor gets inserted into which protein, even in structurally similar proteins with different cofactor occupancies. A majority of metal or bioinorganic cofactor transfer reactions are governed by protein interactions, yet mechanistic detail remains unclear for many metalloproteins in how they pass cofactors from one protein to the next, especially at sites buried within proteins. This applies to one of the most ubiquitous types of cofactors found in bacteria, archaea, and eukaryotes, namely iron sulfur (Fe/S) clusters. In the model eukaryotic organism Saccharomyces cerevisiae, 18 mitochondrial Fe/S cluster (ISC) assembly proteins and 11 cytosolic Fe/S protein assembly (CIA) components create an extensive cellular protein network, which synthesizes, traffics, and inserts Fe/S clusters into target apoproteins. An interesting aspect of the CIA machinery in eukaryotes is that many of the trafficking factors themselves bind more than one Fe/S cluster and it remains poorly understood how the CIA components themselves are matured. For example, Nar1 is peculiar because it has been proposed to be a [4Fe-4S] cluster trafficking mediator of the CIA pathway via a labile [4Fe-4S] cluster and at the same time a target apoprotein that requires a buried, non-transferable [4Fe-4S] cluster. Furthermore, Nar1 is homologous to bacterial and algal [FeFe]-hydrogenases, but doesn’t have the characteristic hydrogenase function. Why a hydrogenase-like protein has evolved as an essential eukaryotic Fe/S cluster trafficking protein remains one of the most challenging questions to address in the Fe/S cluster biogenesis field. The aim of this proposal is to use a combination of in vivo, in vitro, and biophysical studies to assess mechanisms of [4Fe-4S] cluster insertions into Nar1 and to use this information to determine the physiological function of Nar1 in the CIA pathway. S. cerevisiae will be used as a tractable organism to study the function of Nar1 and the importance of partner binding proteins in Nar1 maturation. Details into Nar1 maturation will guide reconstitution reactions involving Fe/S cluster transfer to and from Nar1, quantified via isotopically-enriched Fe/S clusters. Biophysical characterization of native Nar1 via protein-protein interaction assays, spectroscopic Fe/S cluster analyses, and X-ray crystallographic structure determination will also be pursued, in part, through essential collaborations in the SPP. Assigning the molecular function of Nar1 will advance our understanding of the multiple ways that Fe/S clusters can be transferred or inserted into apoproteins. Subsequently, this will also enhance our understanding of how disruptions in Fe/S cofactor trafficking lead to Fe/S protein assembly diseases.