Zitieren Sie bitte immer diesen URN: urn:nbn:de:kobv:b43-628217
Yeast [FeFe]-hydrogenase-like protein Nar1 can bind not only two [4Fe-4S] clusters but also a [2Fe-2S] cluster
- 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,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.…


| Autor*innen: | Joseph J. BraymerORCiD, L. Knauer, J. C. Crack, J. Oltmanns, M. Heghmanns, J. C. Soares, N. E. Le Brun, V. Schünemann, M. Kasanmascheff |
|---|---|
| Dokumenttyp: | Preprint |
| Veröffentlichungsform: | Graue Literatur |
| Sprache: | Englisch |
| Titel des übergeordneten Werkes (Englisch): | bioRxiv |
| Jahr der Erstveröffentlichung: | 2025 |
| Organisationseinheit der BAM: | 1 Analytische Chemie; Referenzmaterialien |
| 1 Analytische Chemie; Referenzmaterialien / 1.8 Umweltanalytik | |
| Veröffentlichende Institution: | Bundesanstalt für Materialforschung und -prüfung (BAM) |
| Erste Seite: | 1 |
| Letzte Seite: | 14 |
| DDC-Klassifikation: | Technik, Medizin, angewandte Wissenschaften / Ingenieurwissenschaften / Sanitär- und Kommunaltechnik; Umwelttechnik |
| Freie Schlagwörter: | Hydrogenase-like proteins; Iron sulfur cluster; Metalloprotein |
| Themenfelder/Aktivitätsfelder der BAM: | Umwelt |
| Umwelt / Biokorrosion | |
| DOI: | 10.1101/2025.03.25.644927 |
| URN: | urn:nbn:de:kobv:b43-628217 |
| Verfügbarkeit des Dokuments: | Datei für die Öffentlichkeit verfügbar ("Open Access") |
| Lizenz (Deutsch): | Creative Commons - CC BY - Namensnennung 4.0 International |
| Datum der Freischaltung: | 02.04.2025 |
| Referierte Publikation: | Nein |
| Schriftenreihen ohne Nummerierung: | Preprints der BAM |


