The Yeast Iron Regulatory Proteins Grx3/4 and Fra2 Form Heterodimeric Complexes Containing a [2Fe-2S] Cluster With Cysteinyl and Histidyl Ligation

Biochemistry. 2009 Oct 13;48(40):9569-81. doi: 10.1021/bi901182w.


The transcription of iron uptake and storage genes in Saccharomyces cerevisiae is primarily regulated by the transcription factor Aft1. Nucleocytoplasmic shuttling of Aft1 is dependent upon mitochondrial Fe-S cluster biosynthesis via a signaling pathway that includes the cytosolic monothiol glutaredoxins (Grx3 and Grx4) and the BolA homologue Fra2. However, the interactions between these proteins and the iron-dependent mechanism by which they control Aft1 localization are unclear. To reconstitute and characterize components of this signaling pathway in vitro, we have overexpressed yeast Fra2 and Grx3/4 in Escherichia coli. We have shown that coexpression of recombinant Fra2 with Grx3 or Grx4 allows purification of a stable [2Fe-2S](2+) cluster-containing Fra2-Grx3 or Fra2-Grx4 heterodimeric complex. Reconstitution of a [2Fe-2S] cluster on Grx3 or Grx4 without Fra2 produces a [2Fe-2S]-bridged homodimer. UV-visible absorption and CD, resonance Raman, EPR, ENDOR, Mossbauer, and EXAFS studies of [2Fe-2S] Grx3/4 homodimers and the [2Fe-2S] Fra2-Grx3/4 heterodimers indicate that inclusion of Fra2 in the Grx3/4 Fe-S complex causes a change in the cluster stability and coordination environment. Taken together, our analytical, spectroscopic, and mutagenesis data indicate that Grx3/4 and Fra2 form a Fe-S-bridged heterodimeric complex with Fe ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. Overall, these results suggest that the ability of the Fra2-Grx3/4 complex to assemble a [2Fe-2S] cluster may act as a signal to control the iron regulon in response to cellular iron status in yeast.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cysteine / genetics
  • Cysteine / metabolism*
  • Dimerization
  • Enzyme Stability / genetics
  • Gene Expression Regulation, Enzymologic
  • Gene Expression Regulation, Fungal
  • Glutaredoxins / biosynthesis
  • Glutaredoxins / chemistry*
  • Glutaredoxins / genetics
  • Histidine / genetics
  • Histidine / metabolism*
  • Intracellular Signaling Peptides and Proteins / chemistry*
  • Intracellular Signaling Peptides and Proteins / genetics
  • Iron-Sulfur Proteins / biosynthesis
  • Iron-Sulfur Proteins / chemistry*
  • Iron-Sulfur Proteins / genetics
  • Ligands
  • Multiprotein Complexes / biosynthesis
  • Multiprotein Complexes / chemistry*
  • Multiprotein Complexes / genetics
  • Mutagenesis, Site-Directed
  • Oxidoreductases / biosynthesis
  • Oxidoreductases / chemistry*
  • Oxidoreductases / genetics
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / biosynthesis
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Signal Transduction / genetics


  • FRA2 protein, S cerevisiae
  • Glutaredoxins
  • Grx4 protein, S cerevisiae
  • Intracellular Signaling Peptides and Proteins
  • Iron-Sulfur Proteins
  • Ligands
  • Multiprotein Complexes
  • Saccharomyces cerevisiae Proteins
  • Histidine
  • Grx3 protein, S cerevisiae
  • Oxidoreductases
  • Cysteine