Studies of iron- sulfur cluster containing regulators of the Rrf2 family

Pellicer Martinez, Maria (2017) Studies of iron- sulfur cluster containing regulators of the Rrf2 family. Doctoral thesis, University of East Anglia.

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Abstract

Members of the Rrf2 superfamily of transcription factors are widespread in
bacteria but their biological functions are largely unknown. The aim of the work described
in this thesis was to uncover the distinct iron-sulfur cluster properties and biological
functions of two uncharacterised members of the Rrf2 family.
RirA (Rhizobial iron regularor A) is a global iron regulator in Rhizobium and many
related α-proteobacteria. Spectroscopic and ESI-MS data for cluster-reconstituted RirA
indicated that the protein binds a [4Fe-4S] cluster cofactor. [4Fe-4S] RirA binds DNA,
consistent with its function as a repressor of expression of genes involved in iron uptake.
Under low iron conditions, [4Fe-4S] RirA undergoes cluster conversion resulting in a
[2Fe-2S] form, which binds DNA with lower affinity. If low iron conditions persist, the
[2Fe-2S] cluster is lost, resulting in a cluster-free (apo) form. This does not bind DNA and
can no longer function as a repressor and so the iron-uptake machinery of the cell is
activated. The data are consistent with RirA functioning as a sensor of iron via both ironsulfur
cluster availability and the fragility of its cluster. The significant sensitivity of RirA
to O2 suggests that even when iron is sufficient, the protein is susceptible to cluster
conversion/loss; the interplay between iron and O2 might be important in vivo.
A new member of the Rrf2 superfamily from Streptomyces, RsrR (Redox sensitive
response Regulator), was also characterised. RsrR is isolated containing a [2Fe-2S] cluster
than undergoes facile redox cycling. This modulates its DNA-binding behaviour: it binds
RsrR-regulated promoter DNA tightly only in its oxidised state. Spectroscopic analyses
revealed that the cluster has properties characteristic of His-coordinated [2Fe-2S] clusters
and His12 was identified through site-directed mutagenesis as a likely cluster ligand.
Substitution of Glu8 also significantly affected the cluster properties suggesting it might
also be a cluster ligand.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Chemistry
Depositing User: Katie Miller
Date Deposited: 29 Jun 2017 10:42
Last Modified: 30 Jun 2018 00:38
URI: https://ueaeprints.uea.ac.uk/id/eprint/63979
DOI:

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