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

Research output: Contribution to journalArticlepeer-review

Sopie P. Bennett, Jason C. Crack, Rita Puglisi, Annalisa Pastore, Nick E. Le Brun

Original languageEnglish
JournalChemical Science
Published15 Nov 2022

Bibliographical note

Funding Information: This work was supported by the Biotechnology and Biological Sciences Research Council through grants BB/S001018/1, BB/T017708/1, BB/R013578/1 (to NLB), and BB/S001832/1 (to AP). Further support was provided by a British Mass Spectrometry Society Research Support Grant (to JCC), and by UEA through the purchase/upgrade of the ESI-MS instrument. Publisher Copyright: © 2022 The Royal Society of Chemistry.

King's Authors


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. Fe2+-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 57Fe and 34S. 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 Fe2+ concentrations. Our data indicate that either Fe2+ or sulfur transfer can occur first, but that the transfer of sulfane sulfur (S0) to IscU must occur first if Zn2+ 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 Fe2+ is introduced.

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