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

Braymer, Joseph, Knauer, Lukas, Crack, Jason, Oltmanns, Jonathan, Heghmanns, Melanie, Soares, Jessica, Le Brun, Nick, Schunemann, Volker and Kasanmascheff, Muge (2025) Yeast [FeFe]-hydrogenase-like protein Nar1 binds a [2Fe-2S] cluster. Chemical Science. ISSN 2041-6520

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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.

Item Type:	Article
Additional Information:	Data availability: The data supporting this article can be found within the text and within the supplementary information (SI). Supplementary information: SI Fig. S1–11 and Tables S1–4, SI Methods. See DOI: https://doi.org/10.1039/d5sc04860e. Funding information: J. J. B. and V. S. acknowledge financial support from the Deutsche Forschungsgemeinschaft (DFG)/German Research Foundation (SPP 1927, BR 5640/1-1, SCHU 1251/17-2). This work was also funded by the DFG under Germany's Excellence Strategy (EXC 2033-390677874-RESOLV) to M. K. J. C. C. and N. L. B. acknowledge funding from the UK's Biotechnology and Biological Sciences Research Council (BB/V006851/1).
Faculty \ School:	Faculty of Science > School of Chemistry, Pharmacy and Pharmacology
UEA Research Groups:	Faculty of Science > Research Groups > Chemistry of Life Processes Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry
Depositing User:	LivePure Connector
Date Deposited:	17 Nov 2025 16:30
Last Modified:	18 Nov 2025 10:30
URI:	https://ueaeprints.uea.ac.uk/id/eprint/101042
DOI:	10.1039/d5sc04860e

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