Glutathione-dependent catalysis is a metabolic adaptation to chemical challenges encountered by all life forms. In the course of evolution, nature optimized numerous mechanisms to use glutathione as ...the most versatile nucleophile for the conversion of a plethora of sulfur-, oxygen- or carbon-containing electrophilic substances.
This comprehensive review summarizes fundamental principles of glutathione catalysis and compares the structures and mechanisms of glutathione-dependent enzymes, including glutathione reductase, glutaredoxins, glutathione peroxidases, peroxiredoxins, glyoxalases 1 and 2, glutathione transferases and MAPEG. Moreover, open mechanistic questions, evolutionary aspects and the physiological relevance of glutathione catalysis are discussed for each enzyme family.
It is surprising how little is known about many glutathione-dependent enzymes, how often reaction geometries and acid–base catalysts are neglected, and how many mechanistic puzzles remain unsolved despite almost a century of research. On the one hand, several enzyme families with non-related protein folds recognize the glutathione moiety of their substrates. On the other hand, the thioredoxin fold is often used for glutathione catalysis. Ancient as well as recent structural changes of this fold did not only significantly alter the reaction mechanism, but also resulted in completely different protein functions.
Glutathione-dependent enzymes are excellent study objects for structure–function relationships and molecular evolution. Notably, in times of systems biology, the outcome of models on glutathione metabolism and redox regulation is more than questionable as long as fundamental enzyme properties are neither studied nor understood. Furthermore, several of the presented mechanisms could have implications for drug development. This article is part of a Special Issue entitled Cellular functions of glutathione.
► Fundamental principles of glutathione catalysis are summarized. ► Mechanisms of enzymes with five non-related protein folds are compared. ► Evolutionary aspects and open mechanistic questions are discussed. ► The physiological relevance of glutathione catalysis is highlighted.
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.
Which parts of the glutathione puzzle are most relevant? Does this explain the high intracellular concentrations of reduced glutathione? How can iron-sulfur cluster biogenesis, oxidative protein folding, or redox signaling occur at high glutathione concentrations? Answers to these questions not only seem to depend on the organism, cell type, and subcellular compartment but also on different ideologies among researchers.
A rational approach to compare the relevance of glutathione-dependent pathways is to combine genetic and quantitative kinetic data. However, there are still many missing pieces and too little is known about the compartment-specific repertoire and concentration of numerous metabolites, substrates, enzymes, and transporters as well as rate constants and enzyme kinetic patterns. Gathering this information might require the development of novel tools but is crucial to address potential kinetic competitions and to decipher uncoupling mechanisms to solve the glutathione puzzle. Antioxid. Redox Signal. 27, 1130-1161.
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.
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.
Green fluorescent protein (GFP) is a powerful tool for studying gene expression, protein localization, protein–protein interactions, calcium concentrations, and redox potentials owing to its ...intrinsic fluorescence. However, GFP not only contains a chromophore but is also tightly folded in a temperature-dependent manner. The latter property of GFP has recently been exploited (1) to characterize the translocase of the outer mitochondrial membrane and (2) to discriminate between protein transport across and into biomembranes in vivo. I therefore suggest that GFP could be a valuable tool for the general analysis of protein transport machineries and pathways in a variety of organisms. Moreover, results from such studies could be important for the interpretation and optimization of classical experiments using GFP tagging.
Genetically encoded hydrogen peroxide (H
O
) sensors, based on fusions between thiol peroxidases and redox-sensitive green fluorescent protein 2 (roGFP2), have dramatically broadened the available ..."toolbox" for monitoring cellular H
O
changes. Recent Advances: Recently developed peroxiredoxin-based probes such as roGFP2-Tsa2ΔC
offer considerably improved H
O
sensitivity compared with previously available genetically encoded sensors and now permit dynamic, real-time, monitoring of changes in endogenous H
O
levels.
The correct understanding and interpretation of probe read-outs is crucial for their meaningful use. We discuss probe mechanisms, potential pitfalls, and best practices for application and interpretation of probe responses and highlight where gaps in our knowledge remain.
The full potential of the newly available sensors remains far from being fully realized and exploited. We discuss how the ability to monitor basal H
O
levels in real time now allows us to re-visit long-held ideas in redox biology such as the response to ischemia-reperfusion and hypoxia-induced reactive oxygen species production. Further, recently proposed circadian cycles of peroxiredoxin hyperoxidation might now be rigorously tested. Beyond their application as H
O
probes, roGFP2-based H
O
sensors hold exciting potential for studying thiol peroxidase mechanisms, inactivation properties, and the impact of post-translational modifications, in vivo. Antioxid. Redox Signal. 29, 552-568.
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
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
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.
Mitochondrial protein import (MPI) is essential for the biogenesis of mitochondria in all eukaryotes. Current models of MPI are predominantly based on experiments with one group of eukaryotes, the ...opisthokonts. Although fascinating genome database-driven hypotheses on the evolution of the MPI machineries have been published, previous experimental research on non-opisthokonts usually focused on the analysis of single pathways or components in, for example, plants and parasites. In this study, we have established the kinetoplastid parasite Leishmania tarentolae as a model organism for the comprehensive analysis of non-opisthokont MPI into all four mitochondrial compartments. We found that opisthokont marker proteins are efficiently imported into isolated L. tarentolae mitochondria. Vice versa, L. tarentolae marker proteins of all compartments are also imported into mitochondria from yeast. The results are remarkable because only a few of the more than 25 classical components of the opisthokont MPI machineries are found in parasite genome databases. Our results demonstrate that different MPI pathways are functionally conserved among eukaryotes despite significant compositional differences of the MPI machineries. Moreover, our model system could lead to the identification of significantly altered or even novel MPI components in non-opisthokonts. Such differences might serve as starting points for drug development against parasitic protists.
The redox state of the host-parasite unit has been hypothesized to play a central role for the fitness of the intraerythrocytic blood stages of the human malaria parasite Plasmodium falciparum. In ...particular, hemoglobinopathies have been suggested to cause a more oxidizing environment, thereby protecting from severe malaria. Here we determined the redox potential of infected wild-type (hemoglobin AA) or sickle trait (hemoglobin AS) erythrocytes using parasite-encoded variants of the redox-sensitive green-fluorescent protein 2 (roGFP2). Our non-invasive roGFP2 single-cell measurements revealed a reducing steady-state redox potential of −304 ± 11 mV for the erythrocyte cytosol during ring-stage development and a rather sudden oxidation to −278 ± 12 mV during trophozoite-stage development around 28 h post invasion. There was no significant difference between wild-type or sickle trait erythrocytes regarding the stage dependence and the detected increase of the redox potential during the intraerythrocytic life cycle. The steady-state redox potential of the parasite cytosol, between −304 and −313 mV, was highly reducing throughout the life cycle. The redox potential in the parasitophorous vacuole at the interface between the secretory pathway and the erythrocyte was −284 ± 10 mV and remained stable during trophozoite-stage development with implications for the export of disulfide-containing proteins. In summary, P. falciparum blood stage development from the late ring to the early trophozoite stage causes a physiological jump in erythrocyte redox potential irrespective of the presence or absence of hemoglobin S.
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•More reducing redox potential in erythrocytes than previously thought•A physiological +30 mV jump in erythrocyte redox potential during P. falciparum development•No difference in redox potentials of infected wild-type or sickle trait erythrocytes•The parasitophorous vacuole is a redox shielded compartment
Glutaredoxins are small proteins of the thioredoxin superfamily that are present throughout life. Most glutaredoxins fall into two major subfamilies. Class I glutaredoxins are glutathione-dependent ...thiol-disulfide oxidoreductases whilst class II glutaredoxins coordinate Fe–S clusters. Class I glutaredoxins are typically dithiol enzymes with two active-site cysteine residues, however, some enzymatically active monothiol glutaredoxins are also known. Whilst both monothiol and dithiol class I glutaredoxins mediate protein deglutathionylation, it is widely claimed that only dithiol glutaredoxins are competent to reduce protein disulfide bonds. In this study, using a combination of yeast ‘viability rescue’, growth, and redox-sensitive GFP-based assays, we show that two different monothiol class I glutaredoxins can each facilitate the reduction of protein disulfide bonds in ribonucleotide reductase, methionine sulfoxide reductase and roGFP2. Our observations thus challenge the generalization of the dithiol mechanism for glutaredoxin catalysis and raise the question of why most class I glutaredoxins have two active-site cysteine residues.
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