Mechanistic studies of iron-sulfur cluster binding Rrf2 transcriptional regulators

Gray, Elizabeth (2024) Mechanistic studies of iron-sulfur cluster binding Rrf2 transcriptional regulators. Doctoral thesis, University of East Anglia.

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Several members of the Rrf2 family of transcriptional regulators utilise an iron-sulfur cofactor that is key to their sensing mechanism and modulation of their DNA binding capabilities. This thesis focused on two members of this family, IscR and RirA.

IscR (iron-sulfur cluster regulator) is a [2Fe-2S]-binding member of the Rrf2 family that regulates the Isc iron-sulfur cluster biogenesis pathway in many bacteria through the presence or absence of its [2Fe-2S] cluster – in its [2Fe-2S]-bound state it acts a repressor of the isc operon, whilst in its cluster-free (apo) form, it has a reduced affinity for DNA. The direct response of the IscR cluster to signals known to cause in vivo de-repression of isc has not been previously investigated in vitro. Here, a new IscR homolog from Yersinia enterocolitica has been characterised. YeIscR binds a [2Fe-2S] cluster that demonstrates cluster-dependent binding to the iscR promoter. In vitro, the [2Fe-2S] cluster was shown to be stable to O2 and reactive oxygen species (ROS), even though these signals have been shown to alleviate E. coli IscR repression in vivo. [2Fe-2S] YeIscR was shown to be sensitive to low iron under aerobic conditions, but cluster degradation relied on a direct
interaction between YeIscR and the chelator, suggesting that, in vivo, cluster degradation is dependent on additional factors under low iron conditions. Importantly, YeIscR demonstrated significant cluster sensitivity to glutathione under aerobic but not anaerobic conditions. This demonstrates a previously unknown mechanism for direct sensing of O2 and ROS by the [2Fe-2S] cluster of IscR and is consistent with in vivo observations of IscR regulation.

RirA (Rhizobial iron regulator A) is a Rrf2 family member that utilises a [4Fe-4S] cluster in its sensing domain. It is proposed that the [4Fe-4S] cluster is coordinated by three cysteine residues, and iron-sensing occurs through a labile fourth iron. Here, N8C and N8D RirA variants were investigated. N8C RirA demonstrated reduced iron-sensing capabilities and rapid conversion from the [4Fe-4S] cluster to the [2Fe-2S] form in the presence of O2. Both N8C and N8D RirA variants were shown to retain DNA-binding capabilities, but N8C RirA exhibited lower affinity for DNA than N8D or wild-type RirA. Overall, the introduction of a fourth ligand stabilised the RirA cluster, demonstrating the importance of the labile cluster iron for the iron sensing function of RirA.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Chemistry
Depositing User: Nicola Veasy
Date Deposited: 26 Jun 2024 10:21
Last Modified: 26 Jun 2024 10:22

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