Yeast [FeFe]-hydrogenase-like protein Nar1 binds a [2Fe–2S] cluster

Nar1 is an essential eukaryotic protein proposed to function as an iron-sulphur (Fe/S) cluster trafficking factor in the cytosolic iron-sulphur protein assembly (CIA) machinery. However, such a role has remained unclear due to difficulties in purifying adequate amounts of cofactor-bound protein. The [FeFe]-hydrogenase-like protein has two conserved binding sites for [4Fe-4S] clusters but does not show hydrogenase activity <i>in vivo</i> due to the lack of an active site [2Fe]_H cofactor. Here, we report a new preparation procedure for Nar1 that facilitated studies by UV-vis, EPR, and Mössbauer spectroscopies, along with native mass spectrometry. Nar1 recombinantly produced in <i>E. coli</i> contained a [4Fe-4S] cluster, bound presumably at site 1, along with an unexpected [2Fe-2S] cluster bound at an unknown site. Fe/S reconstitution reactions installed a second [4Fe-4S] cluster at site 2, leading to protein with up to three Fe/S cofactors. It is proposed that the [2Fe-2S] cluster occupies a cavity in Nar1 that is filled by the [2Fe]_H cofactor in [FeFe]-hydrogenases. Strikingly, two of the Fe/S clusters were rapidly destroyed by molecular oxygen, linking Nar1 oxygen sensitivity <i>in vitro</i> to phenotypes observed previously <i>in vivo</i>. Our biochemical results, therefore, validate a direct link between cellular oxygen concentrations and the functioning of the CIA pathway. These advances also now allow for the pursuit of <i>in vitro</i> Fe/S cluster transfer assays, which will shed light on Fe/S trafficking and insertion by CIA components.