Nebraska Redox Biology Center Educational Portal


Peroxiredoxins (Prxs) are a ubiquitous family of cysteine-dependent thioredoxin fold oxidoreductases [ 1, 2, 3 ]. Prxs are thought to be the major scavengers of H2O2 in cells [ 2 4 ]. Peroxiredoxin genes are present in nearly all previously characterized organisms, suggesting that these enzymes serve important functions conserved throughout evolution [ 2 ]. In addition to antioxidant function, peroxiredoxins are implicated in cell signaling due to their ability to reduce intracellular levels of hydroperoxides and to serve as floodgates of H2O2 signaling, which posits that Prx inactivation enables peroxide-mediated signaling in eukaryotes [ 4, 5, 6, 7 ] There are 136 solved peroxiredoxin protein structure in Protein Data Bank at this time (June 2015).

3D structure of homodimer a Aeropyrum pernix peroxiredoxin.

The mechanism of peroxide detoxification by peroxiredoxin is based on electron flow from NADPH to thioredoxin reductase, than to thioredoxin and from thioredoxin to peroxiredoxin [ 8, 9, 10, 11, 12, 13 ]. Activity of peroxiredoxins may be inhibited by active site cysteine overoxidation. Sulfiredoxins are enzymes which are involved in proxiredoxin reactivation by reducing of redox active cysteine sulfinic acid to sulfenic acid [ 5, 14, 15 ].

2-Cys peroxiredoxin based hydrogen peroxide detoxification pathway.

Yeast Saccharomyces Cerevisiae Peroxiredoxins:

>>gi|6323138|ref|NP_013210.1| Ahp1 cytosolic peroxiredoxin [Saccharomyces cerevisiae]

>gi|6323613|ref|NP_013684.1| Tsa1 cytosolic peroxiredoxin [Saccharomyces cerevisiae]

>gi|6320661|ref|NP_010741.1| Tsa2 cytosolic peroxiredoxin [Saccharomyces cerevisiae]

>gi|6322180|ref|NP_012255.1| Dot5 nuclear peroxiredoxin [Saccharomyces cerevisiae]

>gi|6319407|ref|NP_009489.1| Prx1 mitichondrial peroxiredoxin [Saccharomyces cerevisiae]

Most of peroxiredoxins have TxxC redox motifs in active site in which threonine and cysteine are separated by two other residues [ 16 ]. Proxiredoxins use redox-active cysteines to reduce peroxides and were originally divided into three categories based on the number of cysteinyl residues directly involved in catalysis [ 8, 9, 11, 12, 13 ].

1) 1-Cys Prxs: 1-Cys Prxs have concerved active site cysteine and do not contain a resolving cysteine. Their cysteine sulfenic acid generated on reaction with peroxides is directly reduced by a thiol-containing electron donor such as thioredoxins or glutathione.

2) Typical 2-Cys Prxs: The typical 2-Cys Prxs are the largest class of peroxiredoxins. They contain very conservative active site and resolving cysteines. Typical 2-Cys Prxs are exists as homodimers containing two identical active sites. Redox active site cysteine reducing proxide and form sulfenic acid and attacks the resolving cysteine located in the C terminus of the other subunit. This reaction results in the formation of a stable intersubunit disulfide bond, which is then reduced by thioredoxins and thioredoxin-like proteins.

3) Atypical 2-Cys Prxs: This category of Prxs has the same mechanism as typical 2-Cys Prxs, hovewer, 2-Cys Prxs are structurally monomeric. In these Prxs, active site cysteine and resolving cysteine are located within the same polypeptide. The reaction with peroxide resultion in formation of condensation reaction resulting in formation of sulfenic acid on active site cysteine and subsequent formation of an intramolecular disulfide bond. Atypical 2-Cys Prxs uses thioredoxin as an electron donor for catalitic cycle.

All three Prx classes have the first step in common, in which the active site cysteine attacks the peroxide substrate and is oxidized to a cysteine sulfenic acid. Only the resolution of the cysteine sulfenic acid distinguishes these three Prx classes [ 1, 4 ].

Peroxiredoxin action mechanism. The common step of peroxide reduction involving nucleophilic attack by the active site cysteine and formation of the cysteine sulfenic acid intermediate shared by all Prxs. The mechanism of resolution of the cysteine sulfenic acid is diferent for three classes of peroxiredoxins.

Peroxiredoxin system is a key component of antioxidant system protecting against oxidative stress through its ability to reduce hydroperoxides. The thioredoxin system provides the electrons to thiol-dependent peroxidases to remove reactive oxygen and nitrogen species [ 2, 4, 13 ].

Peroxiredoxins and signaling. Recent stadies shown that peroxiredoxins and glutathione peroxidases (thiol peroxidases) are involved in H2O2 mediated signaling and suggested that the role of thiol peroxidases in oxidative stress defense may be overestimated [ 4, 5, 6, 7 17 ]. They oxidize regulatory and signaling proteins by transferring of oxidative equvalent from peroxide to signaling proteins, resulting in transcriptional responses and signaling programs. The examples, the response to H2O2 was inhibited in Saccharomyces cerevisiae cells lacking multiple thiol peroxidases [ 17 ]. Tsa1 peroxiredoxin is shown to be involved in activation of a stress-activated MAP kinase in Schizosaccharomyces pombe. [ 18 ]. Mammalian ER peroxiredoxin 4 is involved in transferring oxidative equivalents to the proteins in the disulfide bond formation process [ 19 ]. Cytosolic peroxiredoxins 1 and 2 are involved in activation of apoptosis signaling kinase 1 (ASK1)/p38 signaling pathway by peroxide-induced mechanism [ 20 ]. Yeast peroxiredoxin Tpx1 can transfer a redox signal to transcription factor Pap1 at low hydrogen peroxide concentration, whereas higher concentrations of the oxidant inhibit the Tpx1-Pap1 redox pathway through the temporal inactivation of Tpx1 by oxidation of its catalytic cysteine to a sulfinic acid [ 21 ].

A model of redox regulation of gene expression in yeast. Schematic presentation of the current and proposed models of H2O2 mediated signaling. To activate hydroperoxide-dependent gene expression programs, H2O2 initially oxidizes peroxiredoxins (thioredoxin system example), which in turn oxidize transcription factors, kinases and other target proteins in yeast cells. Oxidation of these targets then elicits transcriptional response, redox regulation, signaling pathways and other programs. The model proposes that thiol peroxidases mediate gene expression, whereas a direct interaction between H2O2 and target proteins (dashed arrow) plays a secondary role. The involvement of multiple thiol peroxidases and their regulated interactions with target proteins could explain specificity of the system. Red arrows indicate the direction of electron flow, which is opposite to the direction of thiol peroxidase-mediated oxidative signals.

Measurement of peroxiredoxin activity. There are four major and commonly used assay for peroxiredoxin activity measurement [ 22, 23 ]:

1) Absorbance-based assay in which Prx activity is coupled to oxidation of NADPH via thioredoxin reductase and thioredoxin. NADPH decay can be monitored at 340 nm.

2) Directy monitoring of the changes in Trx fluorescence as Trx becomes oxidized by peroxiredoxin.

3) Measurement of the second order rate constant of Prx reacting with hydrogen peroxide by monitoring the ability of Prx to prevent peroxide from reacting with horseradish peroxidase.

4) Ferrous oxidation-xylenol orange (FOX) assay which directly measures peroxide-dependent oxidation of Fe2+ to Fe3+.

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