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

## TL;DR

This study shows that the essential protein Nar1 can bind multiple iron-sulfur clusters, including a new [2Fe–2S] cluster, and reveals how oxygen affects its function.

## Contribution

A new preparation method for Nar1 enabled discovery of its ability to bind a [2Fe–2S] cluster and provided insights into its oxygen sensitivity.

## Key findings

- Nar1 can bind up to three Fe/S clusters, including a [2Fe–2S] cluster at an unknown site.
- Two Fe/S clusters in Nar1 are rapidly destroyed by molecular oxygen, explaining its oxygen sensitivity.
- The findings link cellular oxygen levels to the functioning of the CIA pathway for Fe/S protein biogenesis.

## Abstract

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 in vivo 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 E. coli 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 in vitro to phenotypes observed previously in vivo. 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 in vitro Fe/S cluster transfer assays, which will shed light on Fe/S trafficking and insertion by CIA components.

Nar1 is an essential iron–sulphur protein biogenesis component in eukaryotes that can bind not only two [4Fe–4S] clusters but also a newly identified [2Fe–2S] cluster.