TY  - INPR
A1  - Braymer, Joseph J.
A1  - Knauer, L.
A1  - Crack, J. C.
A1  - Oltmanns, J.
A1  - Heghmanns, M.
A1  - Soares, J. C.
A1  - Le Brun, N. E.
A1  - Schünemann, V.
A1  - Kasanmascheff, M.
T1  - Yeast [FeFe]-hydrogenase-like protein Nar1 can bind not only two [4Fe-4S] clusters but also a [2Fe-2S] cluster
T2  - bioRxiv
N2  - Nar1 is an essential eukaryotic protein proposed to function as an iron-sulfur (Fe/S) cluster trafficking factor in the cytosolic iron-sulfur 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, one of which is predicted to be a labile site for cluster transfer to downstream targets. 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 three Fe/S cofactors. Strikingly, one [4Fe-4S] cluster was rapidly destroyed by molecular oxygen, potentially linking Nar1 oxygen sensitivity to phenotypes observed previously in vivo. These advances now allow for the pursuit of in vitro Fe/S cluster transfer assays, which will shed light on Fe/S trafficking by CIA components and how they may facilitate the insertion of [4Fe-4S] and potentially [2Fe-2S] clusters into target proteins in the cytosol.
KW  - Iron sulfur cluster
KW  - Hydrogenase-like proteins
KW  - Metalloprotein
PY  - 2025
UR  - https://nbn-resolving.org/urn:nbn:de:kobv:b43-628217
DO  - https://doi.org/10.1101/2025.03.25.644927
SP  - 1
EP  - 14
AN  - OPUS4-62821
LA  - eng
AD  - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany
ER  -