Characterization of human glutaredoxin 2 as iron-sulfur protein: a possible role as redox sensor

Abstract

Human mitochondrial glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase (active site: Cys-Ser-Tyr-Cys) that facilitates the maintenance of mitochondrial redox homeostasis upon induction of apoptosis by oxidative stress. Here, we have characterized Grx2 as an iron-sulfur center-containing member of the thioredoxin fold protein family. Mossbauer spectroscopy revealed the presence of a four cysteine-coordinated nonoxidizable [2Fe-2S]2+ cluster that bridges two Grx2 molecules via two structural Cys residues to form dimeric holo Grx2. Coimmunoprecipitation of radiolabeled iron with Grx2 from human cell lines indicated the presence of the cluster in vivo. The [2Fe-2S]-bridged dimer was enzymatically inactive, but degradation of the cluster and the resulting monomerization of Grx2 activated the protein. Slow degradation under aerobic conditions was prevented by the presence of glutathione, whereas glutathione disulfide as well as one-electron oxidants or reductants promoted monomerization of Grx2. We propose that the iron-sulfur cluster serves as a redox sensor for the activation of Grx2 during conditions of oxidative stress when free radicals are formed and the glutathione pool becomes oxidized.

Fig. 1.

UV-visible spectra of monomeric apo (dashed line) and dimeric holo (straight line) Grx2. Optical spectra of human apo and holo Grx2 after separation by gel filtration. The spectra were normalized to the respective absorbance at 278 nm. Inset: Elution profile of Grx2 separated by Sephadex G-50. Protein elution was recorded at 280 nm (straight line) and elution of the chromophore at 320 nm (dotted line).

Fig. 2.

Identification of a [2Fe-2S] cluster in Grx2 by Mössbauer spectroscopy. Mössbauer spectra of human Grx2 (3.4 mM) recorded at 80 and at 4.2 K with B = 4 T applied perpendicular to the γ-beam, respectively. The solid line in the zero field spectrum is a fit with a symmetric Lorentzian doublet with isomer shift δ = 0.27 mm·s^-1 and quadrupole splitting ΔE_Q = 0.60 mm·s^-1, whereas in the applied field spectrum, a simulation is shown for S = 0 with parameters δ = 0.27 mm·s^-1, ΔE_Q = +0.61 mm·s^-1, asymmetry parameter η = 0.6. These parameters indicate the presence of iron in tetrahedral sulfur coordination with pure valence state (III) and total spin state S = 0 characteristic of a [2Fe-2S]²⁺ cluster.

Fig. 3.

Analysis of the chromophore and secondary structural content of Grx2 by CD spectroscopy. (A) Ellipticity of the Grx2 chromophore in 50 mM sodium phosphate, pH 8/300 mM NaCl. Traces 1, holo Grx2; 2, holo Grx2 treated with 10 mM H₂O₂; 3, holo Grx2 treated with 10 mM dithionite; 4, apo Grx2. (B) Secondary structural content of apo (open circles) and holo (filled circles) Grx2 in 5 mM potassium phosphate, pH 8/100 mM KCl. (Inset) Difference in ellipticity of apo minus holo Grx2. All spectra represent the average of four spectra recorded at 25°C at a scan rate of 1 nm·min^-1 and corrected for the ellipticity of the respective buffer.

Fig. 4.

Kinetics of the chromophore loss of Grx2 upon treatment with various compounds. Holo Grx2 (45 μM [Fe-S] center) in 50 mM sodium phosphate, pH 8/300 mM NaCl was incubated with 2 mM of the indicated compounds at 25°C. The loss of the chromophore was recorded after the decrease in absorbance at 428 nm.

Fig. 5.

Coimmunoprecipitation of iron and Grx2 from two human cell lines. Grx2 was isolated by immunoprecipitation from BL30 and HeLa cells propagated in the presence of the γ-emmitter ⁵⁵Fe. (A) Iron in immunoprecipitates from BL30 and HeLa was quantified from the specific radioactivity, and protein was determined by ELISA. Lanes: 1, immunoprecipitate of mitochondrial Grx2; 2, immunoprecipitate of cytosolic Grx1. (B) Successful precipitation was confirmed by Western blotting. Lanes: 1, Grx1; 2, Grx2; 3 and 4, pure recombinant Grx1 (7 and 10 ng, I) or Grx2 (5 and 10 ng, II); I, anti-Grx1 Western blot; II, anti-Grx2 Western blot.

Fig. 6.

Model of Grx2 activation in vivo. A high GSH/GSSG ratio stabilizes the holo form of Grx2, whereas the [2Fe-2S] cluster is destroyed by oxidative stress leading to activation of Grx2. Active Grx2 can use electrons from both GSH and Trx reductase for the reduction of low molecular mass as well as protein glutathione-mixed disulfides (19) and protects from apoptosis by preventing cytochrome c release (23).