Glutaredoxins catalyze the reduction of disulfides and are key players in redox metabolism and regulation. While important insights were gained regarding the reduction of glutathione disulfide substrates, the mechanism of non-glutathione disulfide reduction remains highly debated. Here we determined the rate constants for the individual redox reactions between PfGrx, a model glutaredoxin from Plasmodium falciparum, and redox-sensitive green fluorescent protein 2 (roGFP2), a model substrate and versatile tool for intracellular redox measurements. We show that the PfGrx-catalyzed oxidation of roGFP2 occurs via a monothiol mechanism and is up to three orders of magnitude faster when roGFP2 and PfGrx are fused. The oxidation kinetics of roGFP2-PfGrx fusion constructs reflect at physiological GSSG concentrations the glutathionylation kinetics of the glutaredoxin moiety, thus allowing intracellular structure-function analysis. Reduction of the roGFP2 disulfide occurs via a monothiol mechanism and involves a ternary complex with GSH and PfGrx. Our study provides the mechanistic basis for understanding roGFP2 redox sensing and challenges previous mechanisms for protein disulfide reduction.

Glutaredoxins are key players in cellular redox homoeostasis and exert a variety of essential functions ranging from glutathione-dependent catalysis to iron metabolism. The exact structure-function relationships and mechanistic differences among glutaredoxins that are active or inactive in standard enzyme assays have so far remained elusive despite numerous kinetic and structural studies. Here, we elucidate the enzymatic mechanism showing that glutaredoxins require two distinct glutathione interaction sites for efficient redox catalysis. The first site interacts with the glutathione moiety of glutathionylated disulfide substrates. The second site activates glutathione as the reducing agent. We propose that the requirement of two distinct glutathione interaction sites for the efficient reduction of glutathionylated disulfide substrates explains the deviating structure-function relationships, activities and substrate preferences of different glutaredoxin subfamilies as well as thioredoxins. Our model also provides crucial insights for the design or optimization of artificial glutaredoxins, transition-state inhibitors and glutaredoxin-coupled redox sensors.

Glutathione metabolism is comparable to a jigsaw puzzle with too many pieces. It is supposed to comprise (i) the reduction of disulfides, hydroperoxides, sulfenic acids, and nitrosothiols, (ii) the detoxification of aldehydes, xenobiotics, and heavy metals, and (iii) the synthesis of eicosanoids, steroids, and iron-sulfur clusters. In addition, glutathione affects oxidative protein folding and redox signaling. Here, I try to provide an overview on the relevance of glutathione-dependent pathways with an emphasis on quantitative data. Recent Advances: Intracellular redox measurements reveal that the cytosol, the nucleus, and mitochondria contain very little glutathione disulfide and that oxidative challenges are rapidly counterbalanced. Genetic approaches suggest that iron metabolism is the centerpiece of the glutathione puzzle in yeast. Furthermore, recent biochemical studies provide novel insights on glutathione transport processes and uncoupling mechanisms.

The thioredoxin fold superfamily is highly diverse and contains many enzymatically active glutathione-dependent thiol-disulfide oxidoreductases, for example glutaredoxins and protein disulfide isomerases. However, many thioredoxin fold proteins remain completely uncharacterized, their cellular function is unknown, and it is unclear if they have a redox-dependent enzymatic activity with glutathione or not. Investigation of enzymatic activity traditionally involved time-consuming in vitro characterization of recombinant proteins, limiting the capacity to study novel mechanisms and structure-function relationships. To accelerate our investigation of glutathione-dependent oxidoreductases, we have developed a high-throughput and semi-quantitative assay in yeast. We combined overexpression of the glutathione transporter OPT1 with genetic fusion constructs between glutathione-dependent oxidoreductases and redox-sensitive green fluorescent protein 2 (roGFP2) to allow the rapid characterization of enzymatic activity with physiological substrates. We show that the kinetics of roGFP2 oxidation by glutathione disulfide correlate well with the in vitro-determined activity of the genetically fused glutaredoxins or mutants thereof. Our assay thus allows direct screening of glutaredoxin activity and rapid investigation of structure-function relationships. We also demonstrate that our assay can be used to monitor roGFP2 oxidation by S-nitrosoglutathione (GSNO). We show that glutaredoxins efficiently catalyze oxidation of roGFP2 by GSNO in both live yeast cells and in vitro. In summary, we have established a novel assay for activity screening and characterization of glutathione-dependent oxidoreductases.

The reduction of bis(2-hydroxyethyl)disulfide (HEDS) by reduced glutathione (GSH) is the most commonly used assay to analyze the presence and properties of enzymatically active glutaredoxins (Grx), a family of central redox proteins in eukaryotes and glutathione-utilizing prokaryotes. Enzymatically active Grx usually prefer glutathionylated disulfide substrates. These are converted via a ping-pong mechanism. Sequential kinetic patterns for the HEDS assay have therefore been puzzling since 1991. Here we established a novel assay and used the model enzyme ScGrx7 from yeast and PfGrx from Plasmodium falciparum to test several possible causes for the sequential kinetics such as pre-enzymatic GSH depletion, simultaneous binding of a glutathionylated substrate and GSH, as well as substrate or product inhibition. Furthermore, we analyzed the non-enzymatic reaction between HEDS and GSH by HPLC and mass spectrometry suggesting that such a reaction is too slow to explain high Grx activities in the assay. The most plausible interpretation of our results is a direct Grx-catalyzed reduction of HEDS. Physiological implications of this alternative mechanism and of the Grx-catalyzed reduction of non-glutathione disulfide substrates are discussed.

Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the real-time assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins.

TgPrx2 represents a recently discovered cytosolic 1-Cys peroxiredoxin (Prx) from the intracellular parasite Toxoplasma gondii. Over-expression of the respective gene confers protection against H(2)O(2), suggesting that the protein possesses peroxidase activity. According to the current nomenclature eukaryotic typical and atypical 2-Cys Prx contain a second conserved resolving cysteine residue whereas 1-Cys Prx work on the basis of a monothiol mechanism. Only a few 1-Cys peroxiredoxins have been biochemically characterized to date. Here we describe the mechanistic characterization of TgPrx2 in vitro, including site directed mutagenesis studies, gel filtration chromatography, and molecular modeling. TgPrx2 has general antioxidant properties as indicated by its ability to protect glutamine synthetase against a dithiothreitol Fe(3+)-catalyzed oxidation system. However, TgPrx2 does not reduce H(2)O(2) nor tert-butyl hydroperoxide at the expense of glutaredoxin, thioredoxin or glutathione. Cys(47) was identified as the active site cysteine residue. Most interestingly, Cys(47) was found to form an intermolecular disulfide with Cys(209) from the C-terminal domain of a second subunit which acts as the resolving cysteine. This is a mechanism analogous to typical peroxiredoxins. In contrast to the latter, however, dimeric TgPrx2 does not oligomerize to decamers but is able to form tetramers and hexamers which are non-covalently associated. To our knowledge, TgPrx2 is the first eukaryotic 'so called' 1-Cys peroxiredoxin shown to act on the basis of a 2-Cys mechanism. Our data indicate that mechanistic studies are essential for classifying peroxiredoxins.