Histidine 103 in Fra2 is an iron-sulfur cluster ligand in the [2Fe-2S] Fra2-Grx3 complex and is required for in vivo iron signaling in yeast.
The BolA homologue Fra2 and the cytosolic monothiol glutaredoxins Grx3 and Grx4 together play a key role in regulating iron homeostasis in Saccharomyces cerevisiae. Genetic studies indicate that Grx3/4 and Fra2 regulate activity of the iron-responsive transcription factors Aft1 and Aft2 in response to mitochondrial Fe-S cluster biosynthesis. We have previously shown that Fra2 and Grx3/4 form a [2Fe-2S](2+)-bridged heterodimeric complex with iron ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. To further characterize this unusual Fe-S-binding complex, site-directed mutagenesis was used to identify specific residues in Fra2 that influence Fe-S cluster binding and regulation of Aft1 activity in vivo. Here, we present spectroscopic evidence that His-103 in Fra2 is an Fe-S cluster ligand in the Fra2-Grx3 complex. Replacement of this residue does not abolish Fe-S cluster binding, but it does lead to a change in cluster coordination and destabilization of the [2Fe-2S] cluster. In vivo genetic studies further confirm that Fra2 His-103 is critical for control of Aft1 activity in response to the cellular iron status. Using CD spectroscopy, we find that ∼1 mol eq of apo-Fra2 binds tightly to the [2Fe-2S] Grx3 homodimer to form the [2Fe-2S] Fra2-Grx3 heterodimer, suggesting a mechanism for formation of the [2Fe-2S] Fra2-Grx3 heterodimer in vivo. Taken together, these results demonstrate that the histidine coordination and stability of the [2Fe-2S] cluster in the Fra2-Grx3 complex are essential for iron regulation in yeast.
Li
Haoran
H
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, USA.
Mapolelo
Daphne T
DT
Dingra
Nin N
NN
Keller
Greg
G
Riggs-Gelasco
Pamela J
PJ
Winge
Dennis R
DR
Johnson
Michael K
MK
Outten
Caryn E
CE
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006639:Histidine; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013057:Spectrum Analysis; D013455:Sulfur / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
C090297:AFT1 protein, S cerevisiae; D001059:Apoproteins; C529266:FRA2 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D006639:Histidine; D013455:Sulfur; D007501:Iron; C545181:Grx3 protein, S cerevisiae; D010088:Oxidoreductases