Native mass spectrometric studies of IscSU reveal a concerted, sulfur-initiated mechanism of iron-sulfur cluster assembly

• Published in: Chemical science (Cambridge) Vol. 14; no. 1; pp. 78 - 95
• Main Authors: Bennett, Sophie P, Crack, Jason C, Puglisi, Rita, Pastore, Annalisa, Le Brun, Nick E
• Format: Journal Article
• Language: English
• Published: England Royal Society of Chemistry 21.12.2022; The Royal Society of Chemistry
• Subjects: Adducts; Assembly; Biosynthesis; Chemistry; Clusters; Iron; Iron 57; Mass spectrometry; Mutation; Proteins; Substitution reactions; Sulfur
• ISSN: 2041-6520; 2041-6539
• DOI: 10.1039/d2sc04169c

Iron-sulfur (Fe-S) clusters are cofactors essential for life. Though the proteins that function in the assembly of Fe-S clusters are well known, details of the molecular mechanism are less well established. The Isc (iron-sulfur cluster) biogenesis apparatus is widespread in bacteria and is the closest homologue to the human system. Mutations in certain components of the human system lead to disease, and so further studies of this system could be important for developing strategies for medical treatments. We have studied two core components of the Isc biogenesis system: IscS, a cysteine desulfurase; and IscU, a scaffold protein on which clusters are built before subsequent transfer onto recipient apo-proteins. Fe 2+ -binding, sulfur transfer, and formation of a [2Fe-2S] was followed by a range of techniques, including time-resolved mass spectrometry, and intermediate and product species were unambiguously identified through isotopic substitution experiments using 57 Fe and 34 S. Under cluster synthesis conditions, sulfur adducts and the [2Fe-2S] cluster product readily accumulated on IscU, but iron adducts (other than the cluster itself) were not observed at physiologically relevant Fe 2+ concentrations. Our data indicate that either Fe 2+ or sulfur transfer can occur first, but that the transfer of sulfane sulfur (S 0 ) to IscU must occur first if Zn 2+ is bound to IscU, suggesting that it is the key step that initiates cluster assembly. Following this, [2Fe-2S] cluster formation is a largely concerted reaction once Fe 2+ is introduced. Time-resolved native mass spectrometry was used to investigate iron-sulfur cluster assembly on IscU. Data revealed a concerted assembly process in which sulfur (S 0 ) transfer must occur first if IscU is in its Zn 2+ -bound form.