Glutathione complexed Fe-S centers

Abstract

Glutathione (γ-glutamyl-cysteinyl-glycine, GSH) is a major thiol-containing peptide with cellular levels of up to 10 mM. (1) Several recent reports have demonstrated glutaredoxins (Grx) to form [Fe(2)S(2)] cluster-bridged dimers, where glutathione provides two exogenous thiol ligands, and have implicated such species in cellular iron sulfur cluster biosynthesis. We report the finding that glutathione alone can coordinate and stabilize an [Fe(2)S(2)] cluster under physiological conditions, with optical, redox, Mössbauer, and NMR characteristics that are consistent with a [Fe(2)S(2)](GS)(4) composition. The Fe-S assembly protein ISU catalyzes formation of [Fe(2)S(2)](GS)(4) from iron and sulfide ions in the presence of glutathione, and the [Fe(2)S(2)] core undergoes reversible exchange between apo ISU and free glutathione.

Figure 1

(Top) A solution of 10 mM GSH, pH 8.6 was mixed with 1 mM FeCl₃, 10 μM of the NifS sulfur-donor protein from T. maritima and 1 mM Cysteine under anaerobic conditions. Following the addition of cysteine, cluster formation was observed by absorbance spectroscopy. (inset: the absorbance change at 415 nm reflecting the formation of the GS⁻-coordinated [Fe₂S₂]²⁺ cluster). (Bottom) Cyclic voltammetric experiments display an irreversible reduction wave around −340 mV (vs. SHE).

Figure 2

Mössbauer spectrum (taken at 212 K) from a 9.3 mM cluster solution in GSH 10 mM (pH 8.6). The solid line corresponds to a quadrupolar interaction characterized by δ = 0.393(1) mm/s and ΔEQ = 0.676(2) mm/s.

Figure 3

Schematic representation of the glutathione complex of [[Fe₂S₂]²⁺(GS⁻)₄]²⁻ showing cysteine α and β protons (C_α, C_β1,2) and glutamate β protons (E_β1,2), as well as ¹H NMR spectra of glutathione (top) and the [[Fe₂S₂]²⁺(GS⁻)₄]²⁻ complex (bottom). For the latter the two cysteine β protons are observed to shift from 2.96 ppm to 3.32 ppm, and from 2.89 ppm to 2.99 ppm, respectively. The cysteine α-proton shift from 3.72 ppm into the water peak at 4.70 ppm. Spectra were obtained from a 1 mM [[Fe₂S₂]²⁺(GS⁻)₄]²⁻ solution in D₂O at 300.1 K, using a Bruker DRX 500 MHz spectrometer.

Figure 4

(Left) The absorbance at 330 nm is observed to decrease when 50 μM holo ISU is incubated with 10 mM GSH in 50 mM Hepes, 100mM NaCl, pH 8.6. Red: holo ISU in the absence of GSH; Blue: holo ISU with 10 mM GSH added; (Right) The absorbance at 330 nm is found to increase when 15 μM apo ISU is incubated with 100 μM [Fe₂S₂](GS)₄ in 50 mM Hepes, 100 mM NaCl, 1 mM DTT, pH 8.6 as a result of cluster transfer to the apo protein.

Scheme 1

Degradation of cluster in the absence of excess glutathione