Significance: 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 Critical Issues: Which p

The Incomplete Glutathione Puzzle: Just Guessing at Numbers and Figures?

Hydrogen peroxide (H2O2) plays important roles in cellular signaling, yet nonetheless is toxic at higher concentrations. Surprisingly, the mechanism(s) of cellular H2O2 toxicity remain poorly understood. Here, we reveal an important role for mitochondrial 1‐Cys peroxiredoxin from budding yeast, Prx1, in regulating H2O2‐induced cell death. We show that Prx1 efficiently transfers oxidative equivalents from H2O2 to the mitochondrial glutathione pool. Deletion of PRX1 abrogates glutathione oxidation and leads to a cytosolic adaptive response involving upregulation of the catalase, Ctt1. Both of these effects contribute to improved cell viability following an acute H2O2 challenge. By replacing PRX1 with natural and engineered peroxiredoxin variants, we could predictably induce widely differing matrix glutathione responses to H2O2. Therefore, we demonstrated a key role for matrix glutathione oxidation in driving H2O2‐induced cell death. Finally, we reveal that hyperoxidation of Prx1 serves as a switch‐off mechanism to limit oxidation of matrix glutathione at high H2O2 concentrations. This enables yeast cells to strike a fine balance between H2O2 removal and limitation of matrix glutathione oxidation.

Hyperoxidation of mitochondrial peroxiredoxin limits H2O2‐induced cell death in yeast

Redox-sensitive green fluorescent protein 2 (roGFP2) is a valuable tool for redox measurements in living cells. Here, we demonstrate that roGFP2 can also be used to gain mechanistic insights into redox catalysis in vivo. In vitro enzyme properties such as the rate-limiting reduction of wild type and mutant forms of the model peroxiredoxin PfAOP are shown to correlate with the ratiometrically measured degree of oxidation of corresponding roGFP2 fusion proteins. Furthermore, stopped-flow kinetic measurements of the oxidative half-reaction of PfAOP support the interpretation that changes in the roGFP2 signal can be used to map hyperoxidation-based inactivation of the attached peroxidase. Potential future applications of our system include the improvement of redox sensors, the estimation of absolute intracellular peroxide concentrations and the in vivo assessment of protein structure-function relationships that cannot easily be addressed with recombinant enzymes, for example, the effect of post-translational protein modifications on enzyme catalysis.

Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells

Aims: Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases involved in oxidant defense and signal transduction. Despite much study, the precise roles of conserved residues remain poorly defined. In this study, we carried out extensive functional and structural characterization of 10 variants of such residues in a model decameric bacterial Prx. Results: Three active site proximal mutations of Salmonella typhimurium AhpC, T43V, R119A, and E49Q, lowered catalytic efficiency with hydrogen peroxide by 4–5 orders of magnitude, but did not affect reactivity toward their reductant, AhpF. pKa values of the peroxidatic cysteine were also shifted up by 1–1.3 pH units for these and a decamer disruption mutant, T77I. Except for the decamer-stabilizing T77V, all mutations destabilized decamers in the reduced form. In the oxidized form, three mutants—T77V, T43A, and T43S—exhibited stabilized decamers and were more efficiently reduced by AhpF than wild-type AhpC. Crystal structures of most mutants wer...

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Experimentally Dissecting the Origins of Peroxiredoxin Catalysis.

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.

Joshua Koduka

Papers

Plasmodium falciparum antioxidant protein reveals a novel mechanism for balancing turnover and inactivation of peroxiredoxins