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

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.

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

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

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

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

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 ΔE_Q = 2.96 mm/s and δ = 1.21 mm/s.

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

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

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

Putative models of the [2Fe-2S] Grx3/4 homodimer (A) and the [2Fe-2S] Fra2-Grx3/4 heterodimer (B). G = GSH.