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

The yeast iron regulatory proteins Grx3/4 and Fra2 form heterodimeric complexes containing a [2Fe-2S] cluster with cysteinyl and histidyl ligation

Haoran Li  1 , Daphne T Mapolelo, Nin N Dingra, Sunil G Naik, Nicholas S Lees, Brian M Hoffman, Pamela J Riggs-Gelasco, Boi Hanh Huynh, Michael K Johnson, Caryn E Outten
Affiliations

The yeast iron regulatory proteins Grx3/4 and Fra2 form heterodimeric complexes containing a [2Fe-2S] cluster with cysteinyl and histidyl ligation

Haoran Li et al. Biochemistry. .

Abstract

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.

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Figures

Figure 1
Figure 1
Fra2 and Grx3/4 copurify as a complex. (A) SDS-PAGE analysis of purified Fra2 and Grx3. Fra2 and Grx3 were individually or co-expressed. NI = non-induced cells, I = induced cells, P = purified protein. The 12–13 kDa band below Fra2 in the purified Fra2 and Grx3-Fra2 lanes is a degradation product of Fra2. The bands shown with an asterisk in the last lane were identified by MALDI-TOF as the E. coli translational elongation factors TufA (43.4 kD) and FusA (77.6 kD). (B) Gel filtration chromatograms of apo (upper chromatogram) and Fe-S forms (bottom chromatogram) of Fra2 and Grx3 (0.5 µg loaded).
Figure 2
Figure 2
UV-visible absorption spectra of as-purified forms of Fe-S cluster-bound Fra2-Grx3 (black line) and Fra2-Grx4 (gray line). ε values are based on Fra2-Grx heterodimer concentrations.
Figure 3
Figure 3
Comparison of the UV-visible absorption and CD spectra of [2Fe-2S] Grx3 (gray line) and [2Fe-2S] Fra2-Grx3 (black line). Spectra were recorded under anaerobic conditions in sealed 0.1 cm cuvettes for [2Fe-2S] Grx3 (0.22 mM in homodimer) in 100 mM Tris-HCl buffer with 250 mM NaCl at pH 7.8 and for [2Fe-2S] Fra2-Grx3 complex (0.18 mM in heterodimer) in 50 mM Tris-MES buffer at pH 8.0. ε and Δε values are based on Grx3 homodimer and Fra2-Grx3 heterodimer concentrations.
Figure 4
Figure 4
Comparison of the resonance Raman spectra of [2Fe-2S] Grx3 and [2Fe-2S] Fra2-Grx3 with 457.9-, 487.9-, and 514.5-nm laser excitation. Samples were ~ 2 mM in [2Fe-2S] cluster and were in the form of a frozen droplet at 17 K. Each spectrum is the sum of 100 scans, with each scan involving photon counting for 1 s at 0.5 cm−1 increments with 6 cm−1 spectral resolution. Bands due to lattice modes of ice have been subtracted from both spectra.
Figure 5
Figure 5
Comparison of the Mössbauer spectra of 57Fe-labeled [2Fe-2S] Grx3 and [2Fe-2S] Fra2-Grx3. Samples were ~ 2 mM in [257Fe-2S] clusters. Spectra were recorded at 4.2 K with a magnetic field of 50 mT applied parallel to the γ-radiation. The solid black lines are theoretical simulations of the [2Fe-2S]2+ cluster spectra using two unresolved equal-intensity doublets, with the parameters listed in Table 3. Contributions from a minor mononuclear high-spin Fe(II) species (10% of total 57Fe for [2Fe-2S] Grx3 and 3% of the total 57Fe for [2Fe-2S] Fra2-Grx3) have been removed from the spectra. Parameters used for the Fe(II) species are ΔEQ = 2.96 mm/s and δ = 1.21 mm/s.
Figure 6
Figure 6
Comparison of the X-band EPR spectra of dithionite-reduced [2Fe-2S] Grx3 and [2Fe-2S] Fra2-Grx3. The samples described in Fig. 3 were reduced under anaerobic conditions by addition of stoichiometric sodium dithionite (i.e. a 2-fold excess of reducing equivalents) and frozen immediately in liquid nitrogen. EPR conditions: microwave frequency, 9.60 GHz; modulation frequency, 100 KHz; modulation amplitude, 0.65 mT; microwave power, 20 mW; temperature, 26 K.
Figure 7
Figure 7
35 GHz CW ENDOR of dithionite-reduced [2Fe-2S] Fra2-Grx3 complex at 2 K, recorded at g = 1.92. The sample was the same as that described in Figure 3, except that it contained 15% (v/v) ethylene glycol, had a [2Fe-2S] cluster concentration of 0.57 mM, and was reduced anaerobically with a 5-fold excess of reducing equivalents from sodium dithionite immediately prior to freezing.
Figure 8
Figure 8
Left Panel: Unfiltered EXAFS data (black) compared to best 3-shell single-scattering fit (fit C from Table 4 in red). Right panel: Fourier transform of the EXAFS data in black and transformed fit C in red.
Figure 9
Figure 9
Putative models of the [2Fe-2S] Grx3/4 homodimer (A) and the [2Fe-2S] Fra2-Grx3/4 heterodimer (B). G = GSH.
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