Molecular mechanism of the reduction of cysteine sulfinic acid of peroxiredoxin to cysteine by mammalian sulfiredoxin

Among many proteins with cysteine sulfinic acid (Cys-SO2H) residues, the sulfinic forms of certain peroxiredoxins (Prxs) are selectively reduced by sulfiredoxin (Srx) in the presence of ATP. All Srx enzymes contain a conserved cysteine residue. To elucidate the mechanism of the Srx-catalyzed reaction, we generated various mutants of Srx and examined their interaction with PrxI, their ATPase activity, and their ability to reduce sulfinic PrxI. Our results suggest that three surface-exposed amino acid residues, corresponding to Arg50, Asp57, and Asp79 of rat Srx, are critical for substrate recognition. The presence of the sulfinic form (but not the reduced form) of PrxI induces the conserved cysteine of Srx to take the gamma-phosphate of ATP and then immediately transfers the phosphate to the sulfinic moiety of PrxI to generate a sulfinic acid phosphoryl ester (Prx-Cys-S(=O)OPO3(2-)). This ester is reductively cleaved by a thiol molecule (RSH) such as GSH, thioredoxin, and dithiothreitol to produce a disulfide-S-monoxide (Prx-Cys-S(=O)-S-R). The disulfide-S-monoxide is further reduced through the oxidation of three thiol equivalents to complete the catalytic cycle and regenerate Prx-Cys-SH.     

Oxidation of cysteine and homocysteine by bovine albumin

The autooxidation of cysteine and homocysteine to their disulfide forms was determined by measuring the time course of thiol groups disappearance. We found the oxidative chemistry of cysteine and homocysteine to be quite different. In the absence of added Cu(II), cysteine autooxidized at a slower rate than homocysteine, though in its presence cysteine oxidation was much faster, homocysteine being found to be a poor responder to copper catalysis. Albumin speeded up the spontaneous oxidation of both aminothiols, the reaction being faster with cysteine than with homocysteine. The copper content of different albumins was found to be highly variable, ranging from 12.75 to 0.64 microg Cu(II)/g albumin. We propose that copper bound to albumin possesses redox cycling activity to perform cysteine oxidation since: (i) copper elimination by copper chelators markedly reduces oxidation; and (ii) a positive correlation exists between the albumin copper content and the oxidation reaction rate.     

Characterization of mammalian sulfiredoxin and its reactivation of hyperoxidized peroxiredoxin through reduction of cysteine sulfinic acid in the active site to cysteine

Peroxiredoxins (Prxs) are a family of peroxidases that reduce hydroperoxides. The cysteine residue in the active site of certain eukaryotic Prx enzymes undergoes reversible oxidation to sulfinic acid (Cys-SO2H) during catalysis, and sulfiredoxin (Srx) has been identified as responsible for reversal of the resulting enzyme inactivation in yeast. We have now characterized mammalian orthologs of yeast Srx with an assay based on monitoring of the reduction of sulfinic Prx by immunoblot analysis with antibodies specific for the sulfinic state. Sulfinic reduction by mammalian Srx was found to be a slow process (kcat = 0.18/min) that requires ATP hydrolysis. ATP could be efficiently replaced by GTP, dATP, or dGTP but not by CTP, UTP, dCTP, or dTTP. Both glutathione and thioredoxin are potential physiological electron donors for the Srx reaction, given that their Km values (1.8 mM and 1.2 microM, respectively) are in the range of their intracellular concentrations, and the Vmax values obtained with the two reductants were similar. Although its pKa is relatively low (approximately 7.3), the active site cysteine of Srx remained reduced even when the active site cysteine of most Prx molecules became oxidized. Finally, depletion of human Srx by RNA interference suggested that Srx is largely responsible for reduction of the Cys-SO2H of Prx in A549 human cells.     

Oxidation of 5-thio-2-nitrobenzoic acid, by the biologically relevant oxidants peroxynitrite anion, hydrogen peroxide and hypochlorous acid.

The modification of protein and non-protein thiols by oxidants including hydrogen peroxide (H(2)O(2)), peroxynitrite anion (ONOO(-)) and hypochlorous acid (HOCl) is well documented. Using an aromatic thiol, 5-thio-2-nitrobenzoic acid, and biologically relevant oxidants, we have identified higher oxidation states of sulfur including the sulfonic acid derivative and the disulfide S-oxide, a thiosulfinate, by HPLC and mass spectrometry. The initial reaction of ONOO(-) with 5-thio-2-nitrobenzoic acid yielded a transient red intermediate, the sulfenate anion. The red intermediate was observed when ONOO(-) and H(2)O(2) were used to oxidize 5-thio-2-nitrobenzoic acid and it persisted for several seconds at pH 7. HOCl oxidized the disulfide, 5,5'dithiobis(2-nitrobenzoic acid) to the corresponding sulfonic acid and no additional products were detected. Using this system, we can directly compare the thiol-oxidizing abilities of several oxidants. Because 5-thio-2-nitrobenzoic acid is the product of the reaction of Ellman's reagent with protein thiols, a detailed study of its stability in biological matrices where oxidants may be generated is warranted.     

Analysis of the oligomeric state of rat 1-Cys peroxiredoxin

Peroxiredoxins (Prxs) are antioxidant proteins with peroxidase and chaperone activities. We assessed the self-association ability of 1-Cys Prx from rat olfactory epithelium. In native PAGE, the recombinant Prx-His₆ produced in E. coli gave two bands corresponding to monomeric (minor) and dimeric (major) protein; incubation of the rPrx with DTT added a third band of oligomers. Western blotting of rat olfactory epithelium proteins resolved by native PAGE (with antiserum to rPrx) revealed that at physiological concentrations the 1-Cys Prx is mostly monomeric and to a lesser extent dimeric. This protein proved highly prone to aggregation in the presence of imidazole.     

Study of the quaternary structure of rat 1-Cys peroxiredoxin

Peroxiredoxins (Prx) are a family of antioxidant proteins with peroxidase activity. The ability of 1-Cys Prx to self-associate was studied with the use of native PAGE and Western blotting. Two protein bands corresponding to monomeric and dimeric forms were detected in the preparation of the recombinant 1-Cys Prx subjected to native PAGE, with dimers being more abundant. The third band corresponding to the oligomeric form was detected after incubation of the recombinant 1-Cys Prx with DTT, although monomers and dimers were also observed. These results indicate that monomeric, dimeric, and oligomeric states of the protein are likely to be interchangeable. Native PAGE in combination with Western blot analysis revealed that self-association of 1-Cys Prx also occurred at physiologically relevant concentrations in vivo. The native 1-Cys Prx existed in the monomeric and dimeric forms in rat olfactory epithelium, with monomers being more common. The structural sensitivity of the recombinant 1-Cys Prx to imidazole was shown.     

Fluorescent and affinity-based tools to detect cysteine sulfenic acid formation in proteins

Cysteine sulfenic acid formation in proteins results from the oxidative modification of susceptible cysteine residues by hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. This species represents a biologically significant modification occurring during oxidant signaling or oxidative stress, and it can modulate protein function. Most methods to identify such oxidatively modified proteins rely on monitoring the loss of one or more thiol group(s) or on selective labeling of nascent thiol groups following reduction of oxidized proteins. Our previous work reported the direct labeling of these chemically distinct modifications with a dimedone analogue, 1,3-cyclohexadione, to which a linker and functional group (an alcohol) had been added; further addition of a fluorescent isatoic acid or methoxycoumarin reporter allowed detection of the incorporated tag by fluorescence techniques ( Poole, L. B., Zeng, B. B., Knaggs, S. A., Yakubu, M., and King, S. B. ( 2005) Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjugate Chem . 16, 1624-1628 ). We have now expanded our arsenal of tagging reagents to include two fluorescein-, two rhodamine-, and three biotin-conjugated probes based on the original approach. The new tools provide readily detectable fluorescent and affinity probes to identify sulfenic acid modifications in proteins and have been used in subsequent mass spectrometric analyses to confirm covalent attachment of the conjugates and directly determine the site of modification.     

Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys peroxide sensor

OhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented by Bacillus subtilis OhrR) and 2-Cys (represented by Xanthomonas campestris OhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. The B. subtilis 1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, the X. campestris 2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-Cys B. subtilis OhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like the X. campestris OhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols.     

Irreversible oxidation of the active-site cysteine of peroxiredoxin to cysteine sulfonic acid for enhanced molecular chaperone activity

The thiol (-SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (-SO(2)H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (-SO(3)H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H(2)O(2) concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO(3)H) by mass spectrometry. Tsa1p-SO(3)H was not an autoxidation product of Tsa1p-SO(2)H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO(3)H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO(3)H multimer lost its peroxidase activity, it gained approximately 4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO(3)H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO(3)H is a marker of cumulative oxidative stress in cells.     

Expression of 1-Cys peroxiredoxin in morphogenic and nonmorphogenic tatar buckwheat calli

Using two-dimensional gel electrophoresis and MALDI-TOF MS, we identified a protein with a mol wt of 24.5 kD and pI = 7.5 as 1-Cys peroxiredoxin. This protein was present among soluble proteins of morphogenic but not nonmorphogenic calli of tatar buckwheat (Fagopyrum tataricum (L.) Gaertn). Its expression was evidently related to the presence of proembryonal cell complexes in morphogenic calli.     

Anti-oxidative stress system in cyanobacteria. Significance of type II peroxiredoxin and the role of 1-Cys peroxiredoxin in Synechocystis sp. strain PCC 6803

Two antioxidant proteins, SLL1621 and SLR1198, were captured in the cyanobacteria Synechocystis sp. PCC 6803 using thioredoxin affinity chromatography, which was first applied to the survey of thioredoxin target proteins in chloroplasts (Motohashi, K., Kondoh, A., Stumpp, M. T., and Hisabori, T. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 11224-11229). They are annotated as AhpC/TSA family protein (SLL1621) and antioxidant protein (SLR1198) in CyanoBase (Nakamura, Y., Kaneko, T., Hirosawa, M., Miyajima, N., and Tabata, S. (1998) Nucleic Acids Res. 26, 63-67). Based on sequence homology analysis SLL1621 and SLR1198 are categorized into type II peroxiredoxin and 1-Cys type peroxiredoxin, respectively. In vitro interaction between SLL1621 and thioredoxin was confirmed using the recombinant proteins expressed in Escherichia coli. Furthermore, we found that SLL1621 shows remarkable glutathione-dependent peroxidase activity. Disruption of the sll1621 gene had a dramatic effect on the viability of the cyanobacterial cells even under weak light conditions (50 micromol.m(-2).s(-1)), suggesting this peroxiredoxin is essential for this cyanobacterium. In contrast, although the peroxidase activity of SLR1198 was scarcely detected, disruption of the gene, slr1198, certainly affected the growth rate of the cells. The results indicate the physiological significance of two different peroxiredoxins as an anti-oxidative stress system in cyanobacteria.     

Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.     

Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1.

Recently several cDNAs have been described encoding lysyl oxidase-like proteins. Their deduced amino acid sequences are characterized by a strong similarity in the C-terminal region, corresponding to the lysyl oxidase family catalytic domain, and by marked differences in the N-terminal regions. Different biological functions have been described for lysyl oxidases in addition to their traditionally assumed cross-linking role. To answer the question of whether these different functions are carried out by different lysyl oxidases, purified and active forms of these enzymes are required. At present only the classical form of lysyl oxidase has been purified and characterized. The purpose of this study was to isolate and characterize the lysyl oxidase-like protein. In view of the strong sequence homology with the C-terminal domain of other lysyl oxidases, we chose to purify the protein from bovine aorta using antibodies specific to the N-terminal domain of the proenzyme. We have isolated a 56-kDa protein identified by amino acid sequencing as the bovine lysyl oxidase-like precursor, which is cleaved at the Arg-Arg-Arg sequence at positions 89-91 by a furin-like activity, as revealed after deblocking of the N-terminal residue. The immunopurified protein was largely inactive, but further processing in vitro by bone morphogenetic protein-1 led to an enzyme that was active on elastin and collagen substrates.     

Homology modeling and docking mechanism of the mercaptosuccinate and methotrexate to P. falciparum 1-Cys peroxiredoxin: a preliminary molecular study

A three-dimensional (3-D) model of 1-Cys peroxiredoxin from P. falciparum (Pf-Prx) has been constructed by homology modeling. The model building was based on a structural alignment with the human 1-Cys peroxiredoxin ray structure. First, mercaptosuccinate was docked by Molecular and Quantum Mechanics at the active site in both isozymes, evidencing the role of different residues in the ligand-protein interaction. Stable conformation of the inhibitor in the active site was obtained from the conformational analysis by molecular dynamics. Next, The complex was reoptimized by semiempirical molecular orbital AM1 method. Conformational and frontier orbitals analyses of the ligand-protein complex were carried out in an attempt to obtain structural insight into the inhibition mechanism. Finally, the docking study of the methotrexate (MTX), an anticancer drug also used as an antimalarial inhibitor, into the modes binding site was performed. From the resulting stable complex structure, it was found that the glutamate ring of MTX fits the active site with high complementarity. The glutamate ring formed two hydrogen bonds to the imidazol group of His41 and the amino groups of Arg129. The side-chain of glutamate was in close proximity to the sulfur atom of the catalytic residue, Cys47. This binding mode suggests a possible inhibition mechanism, whereby the cysteine residue is covered with the glutamate ring of the MTX inhibitor, forming an enzyme-ligand adduct. In addition, the higher interaction energies and the molecular orbitals localization between the Pf-Prx active site and the inhibitors alluded to the probable binding sites of the ligand nucleophilic ring.     

The requirement of reversible cysteine sulfenic acid formation for T cell activation and function

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in mediating cellular responses. We have examined the importance of reversible cysteine sulfenic acid formation in naive CD8(+) T cell activation and proliferation. We observed that, within minutes of T cell activation, naive CD8(+) T cells increased ROI levels in a manner dependent upon Ag concentration. Increased ROI resulted in elevated levels of cysteine sulfenic acid in the total proteome. Analysis of specific proteins revealed that the protein tyrosine phosphatases SHP-1 and SHP-2, as well as actin, underwent increased sulfenic acid modification following stimulation. To examine the contribution of reversible cysteine sulfenic acid formation to T cell activation, increasing concentrations of 5,5-dimethyl-1,3-cyclohexanedione (dimedone), which covalently binds to cysteine sulfenic acid, were added to cultures. Subsequent experiments demonstrated that the reversible formation of cysteine sulfenic acid was critical for ERK1/2 phosphorylation, calcium flux, cell growth, and proliferation of naive CD8(+) and CD4(+) T cells. We also found that TNF-alpha production by effector and memory CD8(+) T cells was more sensitive to the inhibition of reversible cysteine sulfenic acid formation than IFN-gamma. Together, these results demonstrate that reversible cysteine sulfenic acid formation is an important regulatory mechanism by which CD8(+) T cells are able to modulate signaling, proliferation, and function.