Azetidinone sulfenic acids. Isolation of crystalline sulfenic acids from penicillin sulfoxides and a study of their reactivities.
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Crystallization↗
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Synthesis of chemical probes to map sulfenic acid modifications on proteins.
Cysteine sulfenic acids in proteins can be identified by their ability to form adducts with dimedone, but this reagent imparts no spectral or affinity tag for subsequent analyses of such tagged proteins. Given its similar reactivity toward cysteine sulfenic acids, 1,3-cyclohexadione was synthetically modified to an alcohol derivative and linked to fluorophores based on isatoic acid and 7-methoxycoumarin. The resulting compounds retain full reactivity and specificity toward cysteine sulfenic acids in proteins, allowing for incorporation of the fluorescent label into the protein and "tagging" it based on its sulfenic acid redox state. Control experiments using dimedone further show the specificity of the reaction of 1,3-diones with protein sulfenic acids in aqueous media. These new compounds provide the basis for an improved method for the detection of protein sulfenic acids.
Binding Sites↗
Sulfenic acid in human serum albumin.
Sulfenic acid (RSOH) is a central intermediate in both the reversible and irreversible redox modulation by reactive species of an increasing number of proteins involved in signal transduction and enzymatic pathways. In this paper we focus on human serum albumin (HSA), the most abundant plasma protein, proposed to serve antioxidant functions in the vascular compartment. Sulfenic acid in HSA has been previously detected using different methods after oxidation of its single free thiol Cys34 through one- or two-electron mechanisms. Since recent evidence suggests that sulfenic acid in HSA is stabilized within the protein environment, this derivative represents an appropriate model to examine protein sulfenic acid biochemistry, structure and reactivity. Sulfenic acid in HSA could be involved in mixed disufide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments.
Free Radicals↗
Widespread sulfenic acid formation in tissues in response to hydrogen peroxide.
A principal product of the reaction between a protein cysteinyl thiol and hydrogen peroxide is a protein sulfenic acid. Because protein sulfenic acid formation is reversible, it provides a mechanism whereby changes in cellular hydrogen peroxide concentration may directly control protein function. We have developed methods for the detection and purification of proteins oxidized in this way. The methodology is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin-maleimide. Arsenite-dependent signal generation was fully blocked by pretreatment with dimedone, consistent with its reactivity with sulfenic acids to form a covalent adduct that is nonreducible by thiols. The biotin tag facilitates the detection of protein sulfenic acids on Western blots probed with streptavidin-horseradish peroxidase and also their purification by streptavidin-agarose. We have characterized protein sulfenic acid formation in isolated hearts subjected to hydrogen peroxide treatment. We have also purified and identified a number of the proteins that are oxidized in this way by using a proteomic approach. Using Western immunoblotting we demonstrated that a highly significant proportion of some individual proteins (68% of total in one case) form the sulfenic derivative. We conclude that protein sulfenic acids are widespread physiologically relevant posttranslational oxidative modifications that can be detected at basal levels in healthy tissue, and are elevated in response to hydrogen peroxide. These approaches may find widespread utility in the study of oxidative stress, particularly because hydrogen peroxide is used extensively in models of disease or redox signaling.
Animals↗
beta-Sulfinyl alpha,beta-unsaturated carbonyl compounds from enantiomerically pure sulfenic acids.
The addition of enantiopure sulfenic acids to oxoalkynes constitutes a new and efficient methodology for the synthesis of beta-sulfinyl alpha,beta-unsaturated carbonyl compounds. Sulfenic acids 3 and 4 were generated by thermolysis of suitable precursors and trapped in situ by oxoalkynes 5, affording (R(S),E)- and (S(S),E)-3-alkylsulfinyl-1-phenyl-2-propen-1-ones, 4-alkylsulfinyl-3-buten-2-ones, and 3-[(1S)-isoborneol-10-sulfinyl]-2-propenoates 6 and 7 in good yields and in enantiomerically pure form after simple column chromatography. (R(S),E)-3-[(1S)-isoborneol-10-sulfinyl]-1-phenyl-2-propen-1-one (6(R)a) was involved as a heterodiene in inverse-electron-demanding Diels-Alder reactions with readily available electron-rich dienophiles 14 and 15, corroborating in each case the sulfinyl auxiliary capability in controlling the stereochemical outcome of these cycloadditions. Furthermore, the addition of methylmagnesium iodide to the carbonyl moiety of 6(R)a demonstrated that the chiral sulfur atom exerts a remote stereocontrol in this reaction if assisted by the hydroxy group being part of the isoborneol substituent.
Journal Article↗
Human flavin-containing monooxygenase form 2 S-oxygenation: sulfenic acid formation from thioureas and oxidation of glutathione.
Thioureas are oxygenated by flavin-containing monooxygenases (FMOs), forming reactive sulfenic and/or sulfinic acids. Sulfenic acids can reversibly react with GSH and drive oxidative stress through a redox cycle. For this reason, thiourea S-oxygenation is an example of FMO-dependent bioactivation of a xenobiotic. Functional FMO2 is expressed in the lung of 26% of individuals of African descent and 5% of Hispanics but not in Caucasians or Asians. We have previously demonstrated that human FMO2.1 protein expressed in Sf9 microsomes has high activity toward a series of thioureas that are known or suspected lung toxicants including thiourea, 1-phenylthiourea, and ethylenethiourea. We now show by HPLC and LC-MS that 1-phenylthiourea and alpha-naphthylthiourea are converted to their sulfenic acids. GSH in the incubations at concentrations of 0.5-1.0 mM completely eliminated the sulfenic acid with resultant production of GSSG. These results indicate that individuals with the FMO21 allele may be at enhanced risk of pulmonary damage upon exposure to thioureas.
Chromatography, High Pressure Liquid↗
Structure of the native cysteine-sulfenic acid redox center of enterococcal NADH peroxidase refined at 2.8 A resolution.
In order to obtain the crystal structure of the flavoprotein NADH peroxidase with its native Cys42-sulfenic acid redox center, a strategy combining reduced exposure of crystals to ambient oxygen and data collection at -160 degrees C was applied. The structure of the native enzyme to 2.8 A resolution is described; these results conclusively establish the existence of the Cys42-sulfenic acid as the functional non-flavin redox center of the peroxidase and provide the first structure for any naturally occurring protein-sulfenic acid. The Cys42-sulfenic acid atoms C alpha-C beta-S gamma-O roughly define a planar arrangement which is stacked parallel to the si face of the FAD isoalloxazine and positions the sulfenyl oxygen atom only 3.3 A from FAD-C4A. His10-N epsilon 2 contributes a hydrogen bond to the sulfenic acid oxygen, at a distance of 3.2 A. Although one oxygen atom (OX1) of the non-native Cys42-sulfonic acid derivative identified in the earlier wild-type peroxidase structure was taken to represent the native Cys42-sulfenic acid oxygen [Stehle, T., Ahmed, S. A., Claiborne, A., & Schulz, G. E. (1991) J. Mol. Biol. 221, 1325-1344], this structure shows that the sulfenic acid oxygen does not occupy this position, nor is it hydrogen-bonded to Cys42-N as was OX1. Comparison of the native Cys42-sulfenic acid structure with that of two-electron reduced glutathione reductase provides an insight into the sulfenic acid FAD charge-transfer interaction observed with both wild-type and His10 mutant peroxidases. A model of the E.NADH intermediate recently observed in stopped-flow analyses of the enzyme [Crane, E. J., III, Parsonage, D., Poole, L. B., & Claiborne, A. (1995) Biochemistry 34, 14114-14124] has also been generated to assist in analyzing the chemical mechanism of sulfenic acid reduction.
Amino Acid Sequence↗
Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase.
The trapping of a sulfenic acid within the fully active C165S mutant of the AhpC peroxidase protein from Salmonella typhimurium was investigated. The electrophilic reagent employed in these studies, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), has previously been used to modify thiol, amino, and tyrosine hydroxyl groups in proteins; at neutral pH only cysteinyl residues of AhpC proteins are modified. The peroxide-oxidized C165S mutant of AhpC incubated with NBD-Cl gave a product with an absorbance maximum at 347 nm, whereas the thiol-NBD conjugate formed from the reduced protein absorbed maximally at 420 nm. Electrospray ionization mass spectrometry of the modified proteins allowed identification of the species absorbing at 347 nm as a Cys-S(O)-NBD derivative containing one additional oxygen relative to the Cys-S-NBD product. The C165S conjugates with Cys-S(O)-NBD and Cys-S-NBD had no peroxidase activity when compared to unreacted C165S and wild-type AhpC, but were both reactivated through removal of NBD by DTT. Oxidized C165S was also modified by dimedone, a common sulfenic acid reagent, to give the expected inactivated conjugate of higher mass. This reagent was not removed by DTT and blocked any further reaction of the protein with NBD-Cl. NBD modification of Enterococcus faecalis NADH peroxidase, a well-characterized flavoprotein with an active-site sulfenic acid (Cys-SOH), also yielded the spectrally-distinguishable NBD conjugates following incubation of NBD-Cl with oxidized and reduced forms of the denatured peroxidase, indicating a general utility for this reagent with other sulfenic acid-containing proteins. A significant advantage of NBD-Cl over previously-used sulfenic acid reagents such as dimedone is in the retention of the sulfenic acid oxygen in the modified product; differentiation between protein-associated thiols and sulfenic acids is therefore now possible by means of both visible absorbance properties and mass analyses of the NBD-modified proteins.
4-Chloro-7-nitrobenzofurazan↗
13C NMR analysis of the cysteine-sulfenic acid redox center of enterococcal NADH peroxidase.
In order to characterize the native Cys42-sulfenic acid redox center of the flavoprotein NADH peroxidase by NMR, an expression protocol has been developed which yields the [3-13C]Cys42-labeled protein in 100 mg quantities. Difference spectra of the labeled minus unlabeled oxidized enzyme (E) give a peak at 41.3 ppm (relative to dioxane) which represents the Cys42-sulfenic acid. Reduction of labeled E with 1 equiv of NADH gives the air-stable two-electron reduced (EH2) species, and oxidized minus reduced difference spectra give maxima and minima at 41.3 and 30.8 ppm, respectively, corresponding to the Cys42-sulfenic acid and -thiolate species. Peroxide inactivation of E, which has previously been attributed to oxidation of the Cys42-sulfenic acid to the Cys42-sulfinic and/or sulfonic acid states, gives rise to a new maximum in the difference spectrum of Einactive minus E at 57.0 ppm. A similar expression protocol was used to obtain the [ring-2-13C]His-labeled peroxidase HHAA mutant (His10His23Ala87Ala258); the spectral change over the pH range 5.8-7. 8 is attributed to deprotonation of the surface-exposed His23. Furthermore, replacement of Arg303, which is hydrogen bonded to His10, has no effect on the 13C spectrum. These results provide direct evidence in support of the peroxidase Cys42-sulfenic acid/thiol redox cycle and add significantly to our structure-based understanding of protein-sulfenic acid stabilization and function.
Binding Sites↗
Protein-sulfenic acid stabilization and function in enzyme catalysis and gene regulation.
Sulfenic acids (R-SOH) result from the stoichiometric oxidations of thiols with mild oxidants such as H2O2; in solution, however, these derivatives accumulate only transiently due to rapid self-condensation reactions, further oxidations to the sulfinic and/or sulfonic acids, and reactions with nucleophiles such as R-SH. In contrast, oxidations of cysteinyl side chains in proteins, where disulfide bond formation can be prevented and where the reactivity of the nascent cysteine-sulfenic acid (Cys-SOH) can be controlled, have previously been shown to yield stable active-site Cys-SOH derivatives of papain and glyceraldehyde-3-phosphate dehydrogenase. More recently, however, functional Cys-SOH residues have been identified in the native oxidized forms of the FAD-containing NADH peroxidase and NADH oxidase from Streptococcus faecalis; these two proteins constitute a new class within the flavoprotein disulfide reductase family. In addition, Cys-SOH derivatives have been suggested to play important roles in redox regulation of the DNA-binding activities of transcription factors such as Fos and Jun, OxyR, and bovine papillomavirus type 1 E2 protein. Structural inferences for the stabilization of protein-sulfenic acids, drawn from the refined 2.16-A structure of the streptococcal NADH peroxidase, provide a molecular basis for understanding the proposed redox functions of these novel cofactors in both enzyme catalysis and transcriptional regulation.
Catalysis↗
Identification of cysteine sulfenic acid in AhpC of alkyl hydroperoxide reductase.
C165S AhpC in its sulfenate (Cys-SO-) and presumed thiolate (Cys-S-) forms at pH 7 (pKa for sulfenic acid about pH 6.1) exhibit low extinction absorbance bands around 367 and 324 nm, respectively. Sulfenic acid content of the protein can be assessed by its reactivity with the chromophoric TNB anion. Using this technique, H2O2 titrations of C165S AhpC give a maximum of about 1 SOH per subunit on addition of 1.0 to 1.2 equivalents of H2O2. Cys46-SO- is moderately air stable at neutral pH and room temperature and is oxidized at a steady rate of about 10% per half hour. Cys46-SO- of C165S AhpC is reduced in the presence of catalytic amounts of AhpF by approximately 1 equivalent of NADH to regenerate the Cys46-S- species. NBD chloride is extremely useful as a trapping agent for cysteine sulfenic acid. The Cys46-S(O)-NBD adduct absorbs maximally at 347 nm and is 16 amu larger than the Cys46-S-NBD adduct (lambda max = 420 nm) as shown by ESI-MS. Other electrophilic thiol reagents also react with Cys46-SO-; however, iodoacetamide and N-ethylmaleimide reactivities are much lower with Cys46-SO- than with Cys46-S-. These methods are applicable to other sulfenic acid-containing proteins, although in some cases the proteins must be denatured in order to provide accessibility of this species toward labeling agents.
4-Chloro-7-nitrobenzofurazan↗
Reaction of nitric oxide with the free sulfhydryl group of human serum albumin yields a sulfenic acid and nitrous oxide.
Nitric oxide (NO) generated by diethylamine nonoate (DEA/NO), an NO donor, readily oxidized the free sulfhydryl group of human serum albumin (HSA) as well as the sulfhydryl groups of reduced glutathione (GSH) and dithiothreitol (DTT) at pH 7.4 and 37 degrees C. Under anaerobic conditions, the major products of the oxidation of HSA thiol by NO were the sulfenic acid (RSOH) of HSA and nitrous oxide (N2O). The stoichiometry for this reaction, viz., 1 mol of HSA sulfhydryl oxidized to 1 mol of N2O produced, is consistent with a net two-electron oxidation of the protein thiol to a sulfenic acid. The sulfenic acid product of HSA was shown to react with dimedone and GSH, two known reactions of sulfenic acids. In contrast, anaerobic oxidation of GSH and DTT by NO gave a stoichiometry close to the expected ratio of 2:1 (sulfhydryl oxidized to N2O produced) for the oxidation of these thiols to their disulfides and N2O. Under aerobic conditions, significant fractions of the sulfhydryl groups of HSA, GSH, and DTT were oxidized to their respective thionitrites, presumably by N2O3. Thionitrite formation was not observed in the absence of oxygen. The production of HSA-sulfenic acid by NO, as well as by other oxidizing agents such as H2O2 and peroxynitrite, followed by its reaction with circulating GSH or L-Cys may account for the mixed disulfides of HSA observed in plasma.
Humans↗
Synthesis and pH-dependent stability of purine-6-sulfenic acid, a putative reactive metabolite of 6-thiopurine.
Previous studies have shown that 6-thiopurine is metabolically activated by hepatic cytochrome P-450 to an intermediate capable of binding to proteins by a mixed disulfide linkage. The identity of the active metabolite was postulated to be purine-6-sulfenic acid. In the present report, we describe the synthesis of the sulfenic acid derivatives of 6-thiopurine and two structurally similar compounds, 9-methyl-6-thiopurine and 4-mercapto-1H-pyrazolo[3,4-d]-pyrimidine. The unusual pH-dependent stability profiles of these compounds in buffered aqueous media are presented and explained on the basis of a disproportionation mechanism of sulfenic acid decomposition. Studies with radiolabeled purine-6-sulfenic acid demonstrate that this species binds directly to hepatic microsomal protein. These results support the proposed involvement of purine-6-sulfenic acid in the metabolic activation and tissue binding of 6-thiopurine.
Animals↗
Inhibition of cathepsin K by nitric oxide donors: evidence for the formation of mixed disulfides and a sulfenic acid.
The cysteine protease cathepsin K is believed to play a key role in bone resorption as it has collagenolytic activity and is expressed predominantly and in high levels in bone resorbing osteoclast cells. The addition of nitric oxide (NO) and NO donors to osteoclasts in vitro results in a reduction of bone resorption, although the mechanism of this effect is not fully understood. The S-nitroso derivatives of glutathione (GSNO) and N-acetylpenicillamine (SNAP) and the non-thiol NO donors NOR-1 and NOR-3 all inhibited the activity of purified cathepsin K in a time- and concentration-dependent manner (IC(50) values after 15 min of preincubation at pH 7.5 of 28, 105, 0.4, and 10 microM, respectively). Cathepsin K activity in Chinese hamster ovary cells stably transfected with cathepsin K was also inhibited by the above NO donors with similar potencies. GSNO at 100 microM also completely inhibited the autocatalytic maturation at pH 4.0 of procathepsin K to cathepsin K. The inhibition of cathepsin K by GSNO was rapidly reversed by DTT, but inhibition by NOR-1 was not reversed by DTT, and analysis of the inhibited cathepsin K for S-nitrosylation using the Greiss reaction gave negative results in both cases. Analysis of the protein by electrospray liquid chromatography/mass spectrometry showed that the inhibition of cathepsin K by GSNO resulted in a mass increase of 306 +/- 2 Da, consistent with the formation of a glutathione adduct. Prior inhibition of cathepsin K by the active site thiol-modifying inhibitor E-64 blocked the modification by GSNO, indicating that the glutathione adduct is likely formed at the active site cysteine. Treatment of cathepsin K with NOR-1 resulted in a mass increase of between 30 and 50 Da, corresponding to the oxidation of a cysteine to sulfinic and sulfonic acids. Cotreatment of cathepsin K with NOR-1 plus the sulfenic acid reagent dimedone resulted in a mass increase of approximately 141 Da, which is consistent with the formation of a dimedone adduct. This result demonstrates that the NOR-1-dependent formation of cathepsin K sulfinic and sulfonic acids occurs via a sulfenic acid. These results show that inhibition of cathepsin K activity and its autocatalytic maturation represent two potential mechanisms by which NO can exert its inhibitory effect on bone resorption. This work also shows that oxidative thiol modifications besides S-nitrosylation should be considered when the effects of NO and NO donors on critical thiol-containing proteins are investigated.
Animals↗
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.
4-Chloro-7-nitrobenzofurazan↗
Identification and localization of a stable sulfenic acid in peroxide-treated tetrachlorohydroquinone dehalogenase using electrospray mass spectrometry.
BACKGROUND: Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachlorohydroquinone to trichlorohydroquinone and then to 2,6-dichlorohydroquinone. This enzyme undergoes oxidative damage during purification which causes it to form aberrant products. The damage is reversible by treatment with dithiothreitol. Possible types of oxidative damage include an inappropriate disulfide bond, a cysteine sulfenic acid, or a methionine sulfoxide. RESULTS: Using electrospray liquid chromatography / mass spectrometry, we have demonstrated that oxidation of tetrachlorohydroquinone dehalogenase with H2O2 results in formation of a sulfenic acid at Cys13. Further oxidation to a sulfinic acid was also observed. CONCLUSIONS: Oxidation of Cys 13 to a sulfenic acid prevents the normal reductive dehalogenation reaction from being completed. This finding is consistent with previous work which suggested that Cys 13 acts as a nucleophile during the conversion of tetrachlorohydroquinone to trichlorohydroquinone. The technique described for identification and localization of the cysteine sulfenic acid should be applicable to a wide variety of biological systems.
Amino Acid Sequence↗
L-cysteine, a versatile source of sulfenic acids. Synthesis of enantiopure alliin analogues.
[reaction: see text] l-Cysteine is a stimulating starting product for the generation of transient sulfenic acids, such as 4, 6, 9, and 15, which add to suitable acceptors, allowing formation of sulfoxides showing a biologically active residue. These sulfoxides are easily isolated in enantiomerically pure form. For instance, N-(tert-butoxycarbonyl)-l-cysteine methyl ester (1a) furnished in few steps sulfenic acid 9a, which was readily converted into (R,S(S))-(2-tert-butoxycarbonylamino-2-methoxycarbonyl-ethylsulfinyl)ethene (22), the methyl ester of Boc-protected nor-alliin. Moreover, the addition of 9a to 2-methyl-1-buten-3-yne has led to a sulfur epimeric and separable mixture of (R)-2-(2-tert-butoxycarbonylamino-2-methoxycarbonyl-ethylsulfinyl)-3-methyl-buta-1,3-dienes 10a and 11a, still possessing a "masked" sulfenic acid function, producible from their cysteine moieties once the dienes have been converted into the desired derivatives.
Cysteine↗
Reactions of sulfenic acid with 2-mercaptoethanol: a mechanism for the inhibition of gastric (H+-K+)-adenosine triphosphate by omeprazole.
The reactions of omeprazole, a potent proton pump inhibitor (PPI) were investigated in the presence of 2-mercapotoethanol. Reactions were monitored in solutions buffered to pH values ranging 2.0-8.0 using differential pulse polarography (DPP) at the static mercury drop electrode (SMDE). The fast, sensitive and selective electrochemical technique facilitated successive recordings of voltammograms (peak current (nA) vs. peak potential (volts vs. Ag/AgCl)) for all analytes in situe, including the 2-mercaptoethanol. In acidic solutions and in the presence of 2-mercaptoethanol, omeprazole undergoes degradation into three compounds, the first is a cyclic sulfenamide (D+), previously believed to be the active inhibitor of the H+, K+-ATPase, the second is the omeprazole dimer, and the third is the disulfide believed to be the product of reaction between 2-mercaptoethanol and D+. The cyclic sulfenamide (D+) solution was found to be stable in solutions containing 2-mercaptoethanol having pH values: 2.0, 4.0, and 6.0. This finding proved conclusively that the cyclic sulfenamide is not reactive toward the 2-mercaptoethanol. In contrast to previous reports, the conversion of the sulfenic acid intermediate into D+ was found to be irreversible. Due to this irreversibility, D+ and sulfenic acid were not rapidly interconvertable. The present work suggests that the active inhibitor is the sulfenic acid.
Anti-Ulcer Agents↗