Stabilisation of the RirA [4Fe-4S] cluster results in loss of iron-sensing function

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• RirA is a global iron regulator in Alphaproteobacteria, part of the Rrf2 superfamily.
• It utilizes an iron-sulfur (Fe-S) cluster for DNA binding and iron regulation.
• Cluster instability under iron deficiency is crucial for RirA's regulatory function.

Purpose of the Study:

• To investigate the role of the fourth ligand in the RirA Fe-S cluster.
• To explore if stabilizing the Fe-S cluster affects RirA's iron-sensing ability.
• To understand how residue substitution impacts Rrf2 superfamily regulator function.

Main Methods:

• Structural modeling of RirA based on NsrR.
• Site-directed mutagenesis of RirA (Asn8 to Asp or Cys).
• Spectroscopic analysis and in vivo gene repression assays.

Main Results:

• Mutant RirA proteins (N8D, N8C) formed stable [4Fe-4S] clusters.
• Variants retained DNA-binding and repressor activity but showed increased stability.
• Mutants exhibited significantly reduced or abolished iron-sensing capacity.

Conclusions:

• Cluster fragility is essential for RirA's iron-sensing mechanism.
• A single amino acid substitution can alter Fe-S cluster coordination and regulator function.
• This highlights a general principle for Rrf2 superfamily regulators.