Carbon dioxide/bicarbonate is required for sensitive inactivation of mammalian glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contains an active site Cys and is one of the most sensitive cellular enzymes to oxidative inactivation and redox regulation. Here, we show that inactivation by hydrogen peroxide is strongly enhanced in the presence of carbon dioxide/bicarbonate. Inactivation of isolated mammalian GAPDH by H2O2 increased with increasing bicarbonate concentration and was sevenfold faster in 25 mM (physiological) bicarbonate compared with bicarbonate-free buffer of the same pH. H2O2 reacts reversibly with CO2 to form a more reactive oxidant, peroxymonocarbonate (HCO4-), which is most likely responsible for the enhanced inactivation. However, to account for the extent of enhancement, we propose that GAPDH must facilitate formation and/or targeting of HCO4- to promote its own inactivation. Inactivation of intracellular GAPDH was also strongly enhanced by bicarbonate: treatment of Jurkat cells with 20 µM H2O2 in 25 mM bicarbonate buffer for 5 min caused almost complete GAPDH inactivation, but no loss of activity when bicarbonate was not present. H2O2-dependent GAPDH inhibition in bicarbonate buffer was observed even in the presence of reduced peroxiredoxin 2 and there was a significant increase in cellular glyceraldehyde-3-phosphate/dihydroxyacetone phosphate. Our results identify an unrecognized role for bicarbonate in enabling H2O2 to influence inactivation of GAPDH and potentially reroute glucose metabolism from glycolysis to the pentose phosphate pathway and NAPDH production. They also demonstrate what could be wider interplay between CO2 and H2O2 in redox biology and the potential for variations in CO2 metabolism to influence oxidative responses and redox signaling.

Hypothiocyanous acid reductase is critical for host colonization and infection by Streptococcus pneumoniae.

The major human pathogen Streptococcus pneumoniae encounters the immune-derived oxidant hypothiocyanous acid (HOSCN) at sites of colonization and infection. We recently identified the pneumococcal hypothiocyanous acid reductase (Har), a member of the flavoprotein disulfide reductase enzyme family, and showed that it contributes to the HOSCN tolerance of S. pneumoniae in vitro. Here, we demonstrate in mouse models of pneumococcal infection that Har is critical for colonization and invasion. In a colonization model, bacterial load was attenuated dramatically in the nasopharynx when har was deleted in S. pneumoniae. The Δhar strain was also less virulent compared to wild type in an invasion model as reflected by a significant reduction in bacteria in the lungs and no dissemination to the blood and brain. Kinetic measurements with recombinant Har demonstrated that this enzyme reduced HOSCN with near diffusion-limited catalytic efficiency, using either NADH (kcat/KM = 1.2 × 108 M-1s-1) or NADPH (kcat/KM = 2.5 × 107 M-1s-1) as electron donors. We determined the X-ray crystal structure of Har in complex with the FAD cofactor to 1.50 Å resolution, highlighting the active site architecture characteristic for this class of enzymes. Collectively, our results demonstrate that pneumococcal Har is a highly efficient HOSCN reductase, enabling survival against oxidative host immune defenses. In addition, we provide structural insights that may aid the design of Har inhibitors.

Identification of Streptococcus pneumoniae genes associated with hypothiocyanous acid tolerance through genome-wide screening.

Streptococcus pneumoniae is a commensal bacterium and invasive pathogen that causes millions of deaths worldwide. The pneumococcal vaccine offers limited protection, and the rise of antimicrobial resistance will make treatment increasingly challenging, emphasizing the need for new antipneumococcal strategies. One possibility is to target antioxidant defenses to render S. pneumoniae more susceptible to oxidants produced by the immune system. Human peroxidase enzymes will convert bacterial-derived hydrogen peroxide to hypothiocyanous acid (HOSCN) at sites of colonization and infection. Here, we used saturation transposon mutagenesis and deep sequencing to identify genes that enable S. pneumoniae to tolerate HOSCN. We identified 37 genes associated with S. pneumoniae HOSCN tolerance, including genes involved in metabolism, membrane transport, DNA repair, and oxidant detoxification. Single-gene deletion mutants of the identified antioxidant defense genes sodA, spxB, trxA, and ahpD were generated and their ability to survive HOSCN was assessed. With the exception of ΔahpD, all deletion mutants showed significantly greater sensitivity to HOSCN, validating the result of the genome-wide screen. The activity of hypothiocyanous acid reductase or glutathione reductase, known to be important for S. pneumoniae tolerance of HOSCN, was increased in three of the mutants, highlighting the compensatory potential of antioxidant systems. Double deletion of the gene encoding glutathione reductase and sodA sensitized the bacteria significantly more than single deletion. The HOSCN defense systems identified in this study may be viable targets for novel therapeutics against this deadly pathogen. IMPORTANCE Streptococcus pneumoniae is a human pathogen that causes pneumonia, bacteremia, and meningitis. Vaccination provides protection only against a quarter of the known S. pneumoniae serotypes, and the bacterium is rapidly becoming resistant to antibiotics. As such, new treatments are required. One strategy is to sensitize the bacteria to killing by the immune system. In this study, we performed a genome-wide screen to identify genes that help this bacterium resist oxidative stress exerted by the host at sites of colonization and infection. By identifying a number of critical pneumococcal defense mechanisms, our work provides novel targets for antimicrobial therapy.

A newly identified flavoprotein disulfide reductase Har protects Streptococcus pneumoniae against hypothiocyanous acid.

Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced from hydrogen peroxide and thiocyanate anions by heme peroxidases in secretory fluids such as in the human respiratory tract. Some respiratory tract pathogens display tolerance to this oxidant, which suggests that there might be therapeutic value in targeting HOSCN defense mechanisms. However, surprisingly little is known about how bacteria protect themselves from HOSCN. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized flavoprotein disulfide reductase with HOSCN reductase activity, which we term Har (hypothiocyanous acid reductase), in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using deletion mutants, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of GSH import or recycling. Our findings identify a new bacterial HOSCN reductase and demonstrate a role for this protein in combination with GSH utilization to protect S. pneumoniae from HOSCN.

Governance in the implementation of the UN sustainable development goals in higher education: global trends.

Governance is a key component for implementing sustainable development (SD) initiatives in university teaching, research, and projects. This line of thinking also applies to implementing the United Nations (UN) sustainable development goals (SDGs). Despite the role of governance in guiding processes related to the SDGs, few studies have examined these relations in an integrative manner in higher education. To bridge this knowledge gap, this study assesses the connections between governance and implementing the SDGs at higher education institutions (HEIs). Specifically, it relies on two main methods. The first is a bibliometric analysis, where the literature on the topic has been analyzed. The second method uses case studies from a sample of universities. The combined dual approach has identified the extent to which governance issues influence how these organizations perceive and handle the SDGs. The study provides valuable recommendations that may assist HEIs in implementing the SDGs with a due emphasis on governance.

Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2.

Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H2O2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H2O2 reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by ∼100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H2O2. This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete.

COVID-19: the impact of a global crisis on sustainable development teaching.

The COVID-19 pandemic has caused a global crisis, one which also influences the ways sustainability is being taught at universities. This paper undertakes an analysis of the extent to which COVID-19 as a whole and the lockdown it triggered in particular, which has led to the suspension of presence-based teaching in universities worldwide and influenced teaching on matters related to sustainable development. By means of a worldwide survey involving higher education institutions across all continents, the study has identified a number of patterns, trends and problems. The results from the study show that the epidemic has significantly affected teaching practices. The lockdowns have led to a surge in the use of on-line communication tools as a partial replacement to normal lessons. In addition, many faculty teaching sustainability in higher education have strong competencies in digital literacy. The sampled higher education educations have-as a whole-adequate infrastructure to continue to teach during the lockdowns. Finally, the majority of the sample revealed that they miss the interactions via direct face-to-face student engagement, which is deemed as necessary for the effective teaching of sustainability content. The implications of this paper are two-fold. Firstly, it describes how sustainability teaching on sustainable development has been affected by the lockdown. Secondly, it describes some of the solutions deployed to overcome the problem. Finally, the paper outlines the fact that the COVID-19 pandemic may serve the purpose of showing how university teaching on sustainability may be improved in the future, taking more advantage of modern information technologies.

Bicarbonate is essential for protein-tyrosine phosphatase 1B (PTP1B) oxidation and cellular signaling through EGF-triggered phosphorylation cascades.

Protein-tyrosine phosphatases (PTPs) counteract protein tyrosine phosphorylation and cooperate with receptor-tyrosine kinases in the regulation of cell signaling. PTPs need to undergo oxidative inhibition for activation of cellular cascades of protein-tyrosine kinase phosphorylation following growth factor stimulation. It has remained enigmatic how such oxidation can occur in the presence of potent cellular reducing systems. Here, using in vitro biochemical assays with purified, recombinant protein, along with experiments in the adenocarcinoma cell line A431, we discovered that bicarbonate, which reacts with H2O2 to form the more reactive peroxymonocarbonate, potently facilitates H2O2-mediated PTP1B inactivation in the presence of thioredoxin reductase 1 (TrxR1), thioredoxin 1 (Trx1), and peroxiredoxin 2 (Prx2) together with NADPH. The cellular experiments revealed that intracellular bicarbonate proportionally dictates total protein phosphotyrosine levels obtained after stimulation with epidermal growth factor (EGF) and that bicarbonate levels directly correlate with the extent of PTP1B oxidation. In fact, EGF-induced cellular oxidation of PTP1B was completely dependent on the presence of bicarbonate. These results provide a plausible mechanism for PTP inactivation during cell signaling and explain long-standing observations that growth factor responses and protein phosphorylation cascades are intimately linked to the cellular acid-base balance.

Investigating protein thiol chemistry associated with dehydroascorbate, homocysteine and glutathione using mass spectrometry.

RATIONALE: Oxidative stress is an imbalance between reactive free radical oxygen species and antioxidant defenses. Its consequences can lead to numerous pathologies. Regulating oxidative stress is the complex interplay between antioxidant recycling and thiol-containing regulatory proteins. Understanding these regulatory mechanisms is important for preventing onset of oxidative stress. The aim of this study was to investigae S-thiol protein chemistry associated with oxidized vitamin C (dehydroascorbate, DHA), homocysteine (HcySH) and glutathione (GSH) using mass spectrometry. METHODS: Glutaredoxin-1 (Grx-1) was incubated with DHA, with and without GSH and HcySH. Disulfide formation was followed by electrospray ionization mass spectrometry (ESI-MS) of intact proteins and by LC/ESI-MS/MS of peptides from protein tryptic digestions. The mechanism of DHA-mediated S-thiolation was investigated using two synthetic peptides: AcFHACAAK and AcFHACE. Three proteins, i.e. human hemoglobin (HHb), recombinant peroxiredoxin 2 (Prdx2) and Grx-1, were S-homocysteinylated followed by S-transthiolyation with GSH and investigated by ESI-MS and ESI-MS/MS. RESULTS: ESI-MS analysis reveals that DHA mediates disulfide formation and S-thiolation by HcySH as well as GSH of Grx-1. LC/ESI-MS/MS analysis allows identification of Grx-1 S-thiolated cysteine adducts. The mechanism by which DHA mediates S-thiolation of heptapeptide AcFHACAAK is shown to be via initial formation of a thiohemiketal adduct. In addition, ESI-MS of intact proteins shows that GSH can S-transthiolate S-homocysteinylated Grx-1_ HHb and Prdx2. The GS-S-protein adducts over time dominate the ESI-MS spectrum profile. CONCLUSIONS: Mass spectrometry is a unique analytical technique for probing complex reaction mechanisms associated with oxidative stress. Using model proteins, ESI-MS reveals the mechanism of DHA-facilitated S-thiolation, which consists of thiohemiketal formation, disulfide formation or S-thiolation. Furthermore, protein S-thiolation by HcySH can be reversed by reversible GSH thiol exchange. The use of mass spectrometry with in vitro models of protein S-thiolation in oxidative stress may provide significant insight into possible mechanisms of action occurring in vivo.

Thioredoxin reductase 1 and NADPH directly protect protein tyrosine phosphatase 1B from inactivation during H2O2 exposure.

Regulation of growth factor signaling involves reversible inactivation of protein tyrosine phosphatases (PTPs) through the oxidation and reduction of their active site cysteine. However, there is limited mechanistic understanding of these redox events and their co-ordination in the presence of cellular antioxidant networks. Here we investigated interactions between PTP1B and the peroxiredoxin 2 (Prx2)/thioredoxin 1 (Trx1)/thioredoxin reductase 1 (TrxR1) network. We found that Prx2 becomes oxidized in PDGF-treated fibroblasts, but only when TrxR1 has first been inhibited. Using purified proteins, we also found that PTP1B is relatively insensitive to inactivation by H2O2 but found no evidence for a relay mechanism in which Prx2 or Trx1 facilitates PTP1B oxidation. Instead, these proteins prevented PTP1B inactivation by H2O2 Intriguingly, we discovered that TrxR1/NADPH directly protects PTP1B from inactivation when present during the H2O2 exposure. This protection was dependent on the concentration of TrxR1 and independent of Trx1 and Prx2. The protection was blocked by auranofin and required an intact selenocysteine residue in TrxR1. This activity likely involves reduction of the sulfenic acid intermediate form of PTP1B by TrxR1 and is therefore distinct from the previously described reactivation of end-point oxidized PTP1B, which requires both Trx1 and TrxR1. The ability of TrxR1 to directly reduce an oxidized phosphatase is a novel activity that can help explain previously observed increases in PTP1B oxidation and PDGF receptor phosphorylation in TrxR1 knockout cells. The activity of TrxR1 is therefore of potential relevance for understanding the mechanisms of redox regulation of growth factor signaling pathways.

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin.

Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H2O2 and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m(-1) s(-1)) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.

Mechanistic insight into site-restricted monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI.

A key step in the Fanconi anemia (FA) tumor suppressor pathway is the site-specific monoubiquitination of the FANCD2 protein. Genetic studies indicate that this crucial modification requires eight known FA gene products and the E2-conjugating enzyme Ube2t. Here, we minimally reconstitute this monoubiquitination reaction with Ube2t and the FANCL protein, revealing that monoubiquitination is stimulated by a conserved RWD-like domain in FANCL. Furthermore, addition of the FANCI protein enhances monoubiquitination and also restricts it to the in vivo substrate lysine residue on FANCD2. This work therefore establishes a system that provides mechanistic insight into the functions of FANCL and FANCI in the catalysis of FANCD2 monoubiquitination.

Hyperoxidation of peroxiredoxins 2 and 3: rate constants for the reactions of the sulfenic acid of the peroxidatic cysteine.

Typical 2-Cys peroxiredoxins (Prxs) react rapidly with H2O2 to form a sulfenic acid, which then condenses with the resolving cysteine of the adjacent Prx in the homodimer or reacts with another H2O2 to become hyperoxidized. Hyperoxidation inactivates the Prx and is implicated in cell signaling. Prxs vary in susceptibility to hyperoxidation. We determined rate constants for disulfide formation and hyperoxidation for human recombinant Prx2 and Prx3 by analyzing the relative proportions of hyperoxidized and dimeric products using mass spectrometry as a function of H2O2 concentration (in the absence of reductive cycling) and in competition with catalase at a fixed concentration of H2O2. This gave a second order rate constant for hyperoxidation of 12,000 M(-1) s(-1) and a rate constant for disulfide formation of 2 s(-1) for Prx2. A similar hyperoxidation rate constant for Prx3 was measured, but its rate of disulfide formation was ~10-fold higher, making it is more resistant than Prx2 to hyperoxidation. There are two active sites within the homodimer, and at low H2O2 concentrations one site was hyperoxidized and the other present as a disulfide. Prx with two hyperoxidized sites formed progressively at higher H2O2 concentrations. Although the sulfenic acid forms of Prx2 and Prx3 are ~1000-fold less reactive with H2O2 than their active site thiols, they react several orders of magnitude faster than most reduced thiol proteins. This observation has important implications for understanding the mechanism of peroxide sensing in cells.

Hyperoxidized peroxiredoxin 2 interacts with the protein disulfide- isomerase ERp46.

Prx (peroxiredoxin) 2 protects cells from deleterious oxidative damage. It catalyses the breakdown of hydroperoxides through a highly reactive cysteine residue and has been linked to chaperone activity that promotes cell survival under conditions of oxidative stress. It may also be involved in redox signalling by binding to other proteins. In the present study we have searched for binding partners of Prx2 in H2O2-treated Jurkat and human umbilical vein endothelial cells and discovered that the hyperoxidized form selectively co-precipitated with the protein disulfide-isomerase ERp46. Mutant analyses revealed that loss of the peroxidative cysteine residue of Prx2 also facilitated complex formation with ERp46, even without H2O2 treatment, whereas the resolving cysteine residue of Prx2 was indispensible for the interaction to occur. The complex involved a stable non-covalent interaction that was disassociated by the reduction of intramolecular disulfides in ERp46, or by disruption of the decameric structure of hyperoxidized Prx2. This is the first example of a protein interaction dependent on the hyperoxidized status of a Prx.

Model for the exceptional reactivity of peroxiredoxins 2 and 3 with hydrogen peroxide: a kinetic and computational study.

Peroxiredoxins (Prx) are thiol peroxidases that exhibit exceptionally high reactivity toward peroxides, but the chemical basis for this is not well understood. We present strong experimental evidence that two highly conserved arginine residues play a vital role in this activity of human Prx2 and Prx3. Point mutation of either ArgI or ArgII (in Prx3 Arg-123 and Arg-146, which are ∼3-4 Šor ∼6-7 Šaway from the active site peroxidative cysteine (C(p)), respectively) in each case resulted in a 5 orders of magnitude loss in reactivity. A further 2 orders of magnitude decrease in the second-order rate constant was observed for the double arginine mutants of both isoforms, suggesting a cooperative function for these residues. Detailed ab initio theoretical calculations carried out with the high level G4 procedure suggest strong catalytic effects of H-bond-donating functional groups to the C(p) sulfur and the reactive and leaving oxygens of the peroxide in a cooperative manner. Using a guanidinium cation in the calculations to mimic the functional group of arginine, we were able to locate two transition structures that indicate rate enhancements consistent with our experimentally observed rate constants. Our results provide strong evidence for a vital role of ArgI in activating the peroxide that also involves H-bonding to ArgII. This mechanism could explain the exceptional reactivity of peroxiredoxins toward H(2)O(2) and may have wider implications for protein thiol reactivity toward peroxides.

Genome-wide impact of hydrogen peroxide on maintenance DNA methylation in replicating cells.

BACKGROUND: Environmental factors, such as oxidative stress, have the potential to modify the epigenetic landscape of cells. We have previously shown that DNA methyltransferase (DNMT) activity can be inhibited by sublethal doses of hydrogen peroxide (H2O2). However, site-specific changes in DNA methylation and the reversibility of any changes have not been explored. Using bead chip array technology, differential methylation was assessed in Jurkat T-lymphoma cells following exposure to H2O2. RESULTS: Sublethal H2O2 exposure was associated with an initial genome-wide decrease in DNA methylation in replicating cells, which was largely corrected 72 h later. However, some alterations were conserved through subsequent cycles of cell division. Significant changes to the variability of DNA methylation were also observed both globally and at the site-specific level. CONCLUSIONS: This research indicates that increased exposure to H2O2 can result in long-term alterations to DNA methylation patterns, providing a mechanism for environmental factors to have prolonged impact on gene expression.

The vertebrate Hef ortholog is a component of the Fanconi anemia tumor-suppressor pathway.

The helicase-associated endonuclease for fork-structured DNA (Hef) is an archaeabacterial protein that processes blocked replication forks. Here we have isolated the vertebrate Hef ortholog and investigated its molecular function. Disruption of this gene in chicken DT40 cells results in genomic instability and sensitivity to DNA cross-links. The similarity of this phenotype to that of cells lacking the Fanconi anemia-related (FA) tumor-suppressor genes led us to investigate whether Hef functions in this pathway. Indeed, we found a genetic interaction between the FANCC and Hef genes. In addition, Hef is a component of the FA nuclear protein complex that facilitates its DNA damage-inducible chromatin localization and the monoubiquitination of the FA protein FANCD2. Notably, Hef interacts directly with DNA structures that are intermediates in DNA replication. This discovery sheds light on the origins, regulation and molecular function of the FA tumor-suppressor pathway in the maintenance of genome stability.

Enhanced hyperoxidation of peroxiredoxin 2 and peroxiredoxin 3 in the presence of bicarbonate/CO2.

Hydrogen peroxide undergoes an equilibrium reaction with bicarbonate/CO2 to produce peroxymonocarbonate (HCO4-). Peroxymonocarbonate is more reactive with thiols than H2O2 but it makes up only a small fraction of the H2O2 in physiological bicarbonate buffers so the increase in rate of oxidation of low molecular weight thiols is modest. However, for some thiol proteins such as protein tyrosine phosphatases, the rate enhancement is very much greater. We have investigated the effect of bicarbonate/CO2 on the oxidation of peroxiredoxins (Prdxs) 2 and 3. Using an assay in which reduced Prdx2 inhibits oxidation of horseradish peroxidase by H2O2, we saw no difference between phosphate and bicarbonate buffers (pH 7.4). However, hyperoxidation of both Prdxs in bicarbonate was considerably enhanced. Hyperoxidation involves the reaction of the sulfenic acid formed at the active site with a second H2O2, and prevents its condensation to a disulfide. Using LC/MS analysis, we determined that the presence of 25 mM bicarbonate/CO2 increased the ratio of hyperoxidation compared with condensation 6-fold for Prdx2 and 11-fold for Prdx3. These results imply that Prdx hyperoxidation will occur more readily under physiological conditions than appreciated from in vitro experiments, which seldom use bicarbonate buffers. They also raise the possibility that variations in bicarbonate concentration could provide a mechanism for regulating the cellular level of active Prdxs.

Peroxiredoxin expression and redox status in neutrophils and HL-60 cells.

Peroxiredoxins (Prxs) are thiol peroxidases with a key role in antioxidant defense and redox signaling. They could be important in neutrophils for handling the large amount of oxidants that these cells produce. We investigated the redox state of Prx1 and Prx2 in HL-60 promyelocytic cells differentiated to neutrophil-like cells (dHL-60) and in human neutrophils. HL-60 cell differentiation with dimethyl sulfoxide caused a large decrease in expression of both Prxs, and all-trans retinoic acid also decreased Prx1 expression. Prx1 was mostly reduced in dHL-60 cells. NADPH oxidase activation by phorbol myristate acetate (PMA) or ingestion of Staphylococcus aureus induced rapid oxidation to disulfide-linked dimers, and eventually hyperoxidation. The NADPH oxidase inhibitor, diphenyleneiodonium, prevented Prx1 dimerization in stimulated dHL-60 cells, and decreased the extent of oxidation under resting conditions. In contrast, Prx1 and Prx2 were present in neutrophils from human blood as disulfides, and PMA or S. aureus caused no further oxidation. They remained oxidized on incubation with diphenyleneiodonium in media. Although this suggests that Prx redox cycling could be deficient in neutrophils, thioredoxin expression and thioredoxin reductase activity were similar in neutrophils and dHL-60 cells. Additionally, neutrophil thioredoxin was initially reduced and underwent oxidation after PMA activation. Thus, although the Prxs respond to oxidant generation in dHL-60 cells, in neutrophils they appear "locked" as disulfides. On this basis we propose that neutrophil Prxs are inefficient antioxidants and contribute little to peroxide removal during the oxidative burst, and speculate that they might be involved in other cell processes.