The dithiol mechanism of class I glutaredoxins promotes specificity for glutathione as a reducing agent

Abstract

Class I glutaredoxins reversibly reduce glutathione- and nonglutathione disulfides with the help of reduced glutathione (GSH) using either a monothiol mechanism or a dithiol mechanism. The monothiol mechanism exclusively involves a single glutathionylated active-site cysteinyl residue, whereas the dithiol mechanism requires the additional formation of an intramolecular disulfide bond between the active-site cysteinyl residue and a resolving cysteinyl residue. While the oxidation of glutaredoxins by glutathione disulfide substrates has been extensively characterized, the enzyme-substrate interactions for the reduction of S-glutathionylated glutaredoxins or intramolecular glutaredoxin disulfides are still poorly characterized. Here we compared the thiol-specificity for the reduction of S-glutathionylated glutaredoxins and the intramolecular glutaredoxin disulfide. We show that S-glutathionylated glutaredoxins rapidly react with a plethora of thiols and that the 2nd glutathione-interaction site of class I glutaredoxins lacks specificity for GSH as a reducing agent. In contrast, the slower reduction of the partially strained intramolecular glutaredoxin disulfide involves specific interactions with both carboxylate groups of GSH at the 1st glutathione-interaction site. Thus, the dithiol mechanism of class I glutaredoxins promotes specificity for GSH as a reducing agent, which might explain the prevalence of dithiol glutaredoxins in pro- and eukaryotes.

Keywords: Disulfide; Dithiol; Enzyme mechanism; Glutaredoxin; Glutathione; Redox catalysis; Stopped-flow kinetics.

Graphical abstract

Fig. 1

Catalytic mechanism of class I glutaredoxins. A) Dithiol mechanism for the reversible reduction of nonglutathione disulfides. B) Monothiol mechanism for the reversible reduction of glutathionylated substrates. C) Top view of a model of monothiol class I ScGrx7 with glutathione (GS) at the 1st glutathione-interaction site (left). Residues that were shown to contribute to the 1st glutathione-interaction site are highlighted in salmon (middle). Residues and GS that were shown to form the or to recruit GSH to the transient 2nd glutathione-interaction site are shown in blue (right). D) Schematic representation of a side view of the two distinct glutathione-interaction sites that bind or rather encounter the two glutathione moieties during both half-reactions. E) The dithiol mechanism could resolve sterically blocked enzyme intermediates and reintroduce dead-end species in the catalytic cycle. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Reductive half-reaction between glutathionylated monothiol class I glutaredoxins and various low-molecular-weight thiols. A) Schematic representation of the predicted transition state of the reductive half-reaction. B) Structures and color coding of the tested thiols. C) Representative monophasic stopped-flow reduction kinetics for the reaction between 1 μM S-glutathionylated monothiol PfGrx^{E28W/C32S/C88S} and variable concentrations of GSH. D) Secondary plot for the k_obs values from the single exponential fits for GSH from panel C. E) Brønsted plot of the second-order rate constants from panel D and Fig. S1 (squares) and normalized, pH-independent second-order rate constants (circles). The nucleophile Brønsted coefficient β_nuc was determined from the slope of the linear fit. F–H) Monophasic stopped-flow reduction kinetics, secondary plot, and Brønsted plot for ScGrx7^G107W as in panels C–E. Kinetic traces and secondary plots for the other thiols are shown in Fig. S1 and Fig. S2. All data sets were generated from at least three independent biological replicates with three technical replicates. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

GSSCys assay for wild-type ScGrx7 and ScGrx7^E147Kwith GSH or GABA-SH. A) Structures and color coding of GSH and GABA-SH. B) Schematic representations of the predicted transition state of the reductive half-reaction for the S-glutathionylated enzyme (left) and the inhibited reduced enzyme during the oxidative half-reaction (right). C) Michaelis-Menten plot (left) and Lineweaver-Burk plot (right) for wild-type ScGrx7 and variable concentrations of GSH at 25, 50, and 100 μM GSSCys (shown in lilac, light blue, and dark blue, respectively). D) Michaelis-Menten plot (left) and Lineweaver-Burk plot (right) for wild-type ScGrx7 and variable concentrations of GABA-SH at 50, 100, and 150 μM GSSCys (shown in rose, bright red, and dark red, respectively). Squares were omitted for regression analysis. E) Second-order rate constants from panels C and D calculated form averaged k_cat^app/K_m^app values (top) or reciprocal slopes from secondary plots in Fig. S3 (bottom). F) Lineweaver-Burk plot for ScGrx7^E147K and variable concentrations of GSH at 25, 50, and 100 μM GSSCys. G) Michaelis-Menten plot (left) and Lineweaver-Burk plot (right) for ScGrx7^E147K and variable concentrations of GABA-SH at 50, 100, and 150 μM GSSCys. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Reductive half-reaction between PfGrx^E28W/C88Sdisulfide and various low-molecular-weight thiols. A) Schematic representation of the predicted first transition state of the reductive half-reaction. B) Structures and color coding of the tested thiols. C–H) Secondary plots of the k_obs values for the indicated thiols from the stopped-flow reduction kinetics in Fig. S5. All data sets were generated from at least three independent biological replicates with three technical replicates. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Correlations between the thiol structures and second-order rate constants for PfGrx^E28W/C88Sdisulfide reduction. A) Schematic representation of the predicted first transition state of the reductive half-reaction (top) and structures of the tested thiols with relevant functional groups highlighted (bottom). B) Representative structure of S-glutathionylated ScGrx2^C30S (PDB entry 3D5J) [30] with enzyme-glutathione interactions highlighted. C) Apparent second-order rate constants from Fig. 4 (top) and calculated pH-independent second-order rate constants (bottom) reveal crucial roles of the γ-glutamyl and glycyl carboxylate groups. P values from one way Welch-ANOVA analyses were calculated in R 4.4.1 (P ≤ 0.01: ∗∗; P ≤ 0.001: ∗∗∗).

Fig. 6

Correlation between the ΔG^‡values for PfGrx^E28W/C88Sdisulfide reduction and the thermodynamic stability of mixed glutaredoxin disulfides. A) Structures and color coding of the tested thiols. B) Schematic representation of the predicted first transition state of the reductive half-reaction. C) Circular dichroism spectra of reduced and diamide-oxidized PfGrx^E28W/C88S. D) Correlation between the ΔG^‡ values derived from the second-order rate constants from Fig. 5 and the Gibbs free energy contribution ΔΔG of the mixed disulfides of EcGrx3^C14S/C65Y from Ref. [28]. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Differences in substrate specificity and reaction mechanism for monothiol and dithiol glutaredoxins. A) S-glutathionylated glutaredoxins react with various thiols at the 2nd glutathione-interaction site. Formation of the intramolecular glutaredoxin disulfide (side-reaction) enables the utilization of the highly GSH-specific 1st glutathione-interaction site. B) The rapid reaction of the intramolecular glutaredoxin disulfide with GSH is facilitated by a conformational strain of the disulfide bond and specific enzyme-substrate interactions at the 1st glutathione-interaction site. C) The rapid reaction of S-glutathionylated glutaredoxins with various thiols is facilitated by the low pK_a value of the leaving group (resulting in an asymmetric transition state) and efficient substrate recruitment to the 2nd glutathione-interaction site.