Sulfenic acids as reactive intermediates in xenobiotic metabolism

Sulfenic acid reactive intermediates are formed during the oxidation of cysteine residues of proteins and play key roles in enzyme catalysis, redox homeostasis and regulation of cell signalling. However few data are presently available on the formation and fate of sulfenic acids as reactive intermediates during the metabolism of xenobiotics. This article is a review of the xenobiotic metabolism situations in which the intermediate formation of a sulfenic acid has been reported. Formation of these intermediates has been either proposed on the basis of the isolation of products possibly deriving from sulfenic acids or shown after trapping of the sulfenic acid by specific nucleophiles. This review indicates the different mechanisms by which a sulphur-containing xenobiotic can be metabolized with the intermediate formation of a sulfenic acid. It also indicates the different possible fates of these sulfenic acids that have been reported in the literature. Finally, it discusses the possible implications of the formation of xenobiotic-derived sulfenic acid reactive metabolites in pharmacology and toxicology.     

S-Oxygenation of N-substituted thioureas catalyzed by the pig liver microsomal FAD-containing monooxygenase

The microsomal FAD-containing monooxygenase (EC 1.14.13.8, dimethylaniline monooxygenase) purified to homogeneity from hog liver catalyzes NADPH- and oxygen-dependent S-oxygenation of phenylthiourea, ethylenethiourea, thiocarbanilide, N-methylthiourea, and thiourea to their corresponding formamidine sulfinic acids. The sulfinic acids are formed by sequential enzymic oxidation of the thioureas through intermediate sulfenic acids. The reaction sequence was established by separating intermediate and final oxygenated metabolites of phenylthiourea and ethylenethiourea. The sulfenic and sulfinic acids of these two thioureas, produced enzymically, were chromatographically and spectrally identical with chemically synthesized reference compounds. Phenylformamidine and ethyleneformamidine sulfinic acids are slowly converted to their sulfonic acids upon prolonged incubation. While N-substituted formamidine sulfinic acids oxidize spontaneously to formamidine sulfonic acids at 37 °C, the further oxidation of ethyleneformamidine sulfinic acid may be, at least in part, enzyme catalyzed. The purified monooxygenase also catalyzes rapid oxygenation of mercaptoimidazoles to the corresponding imidazole sulfinic acids. The instability of S-oxygenated mercaptoimidazoles prevented their isolation and positive identification, but analysis of kinetic data obtained with sulfenic acid trapping agents suggests that these compounds are oxygenated by the same reaction sequence established for N-substituted thioureas. The NADPH- and oxygen-dependent oxidation of thiocarbamates and of 2-mercaptoimidazoles catalyzed by hog or hamster liver microsomes correlates with dimethylaniline N-oxidase activity and appears completely independent from cytochrome P-450. The S-oxidation of thiourea and its derivatives is not inhibited by n-octylamine, a known inhibitor of cytochrome P-450 dependent oxygenations. Furthermore, differential thermal inactivation of the flavin-containing monooxygenase totally abolishes phenylthiourea S-oxidase activity of hamster liver microsomes.     

The Redox Biochemistry of Protein Sulfenylation and Sulfinylation

Controlled generation of reactive oxygen species orchestrates numerous physiological signaling events (Finkel, T. (2011) Signal transduction by reactive oxygen species. J. Cell Biol. 194, 7–15). A major cellular target of reactive oxygen species is the thiol side chain (RSH) of Cys, which may assume a wide range of oxidation states (i.e. −2 to +4). Within this context, Cys sulfenic (Cys-SOH) and sulfinic (Cys-SO₂H) acids have emerged as important mechanisms for regulation of protein function. Although this area has been under investigation for over a decade, the scope and biological role of sulfenic/sulfinic acid modifications have been recently expanded with the introduction of new tools for monitoring cysteine oxidation in vitro and directly in cells. This minireview discusses selected recent examples of protein sulfenylation and sulfinylation from the literature, highlighting the role of these post-translational modifications in cell signaling.     

Protein sulfenic acid formation: from cellular damage to redox regulation

Protein sulfenic acid formation has long been regarded as unwanted damage caused by reactive oxygen species (ROS). However, over the past 10 years, accumulating evidence has shown that the reversible oxidation of cysteine thiol groups to sulfenic acid functions as a redox-based signal transduction mechanism. Here, we review the mechanisms of sulfenic acid formation by ROS. We present some of the most important roles played by sulfenic acids in living cells as well as the pathways that regulate sulfenic acid formation. We highlight the experimental tools that have been developed to study the cellular sulfenome and show how computational approaches might help to better understand the mechanisms of sulfenic acid formation.     

Hypothesis: the role of reactive sulfur species in oxidative stress.

Oxidative stress arises from an imbalance in the metabolism of redox-active species promoting the formation of oxidizing agents. At present, these species are thought to include reactive oxygen, reactive nitrogen, and reactive nitrogen oxygen species (ROS, RNS, and RNOS, respectively). Reactive species have their origin in enzymatic synthesis, environmental induction, or by the further chemical reaction of an active species with other endogenous molecules to generate a second-generation reactive species. These second-generation species possess a different spectrum of activity to the parent species, with different redox reactions and biological targets. We now propose that an additional group of redox active molecules termed "reactive sulfur species" (RSS) are formed in vivo under conditions of oxidative stress. RSS are likely to include disulfide-S-oxides, sulfenic acids, and thiyl radicals, and are predicted to modulate the redox status of biological thiols and disulfides.     

The thiol pool in human plasma: The central contribution of albumin to redox processes

The plasma compartment has particular features regarding the nature and concentration of low and high molecular weight thiols and oxidized derivatives. Plasma is relatively poor in thiol-based antioxidants; thiols are in lower concentrations than in cells and mostly oxidized. The different thiol-disulfide pairs are not in equilibrium and the steady-state concentrations of total thiols as well as reduced versus oxidized ratios are maintained by kinetic barriers, including the rates of reactions and transport processes. The single thiol of human serum albumin (HSA-SH) is the most abundant plasma thiol. It is an important target for oxidants and electrophiles due to its reactivity with a wide variety of species and its relatively high concentration. A relatively stable sulfenic (HSA-SO₃H) acid can be formed in albumin exposed to oxidants. Plasma increases in mixed disulfides (HSA-SSR) or in sulfinic (HSA-SO₂H) and sulfonic (HSA-SO₃H) acids are associated with different pathologies and may constitute biomarkers of the antioxidant role of the albumin thiol. In this work we provide a critical review of the plasma thiol pool with a focus on human serum albumin.     

Novel oxidative modifications in redox-active cysteine residues

Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel low-abundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MODi and MODmap algorithm. We observed unexpected mass shifts (Δm=-16, -34, +64, +87, and +103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of -16, -34, and +64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO2-SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO2-Cys) either to DHA (Δm=-34 Da) or serine along with Cys-SO2-SH (Δm=+64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO2-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.     

Nonequilibrium thermodynamics of thiol/disulfide redox systems: a perspective on redox systems biology

Understanding the dynamics of redox elements in biologic systems remains a major challenge for redox signaling and oxidative stress research. Central redox elements include evolutionarily conserved subsets of cysteines and methionines of proteins which function as sulfur switches and labile reactive oxygen species (ROS) and reactive nitrogen species (RNS) which function in redox signaling. The sulfur switches depend on redox environments in which rates of oxidation are balanced with rates of reduction through the thioredoxins, glutathione/glutathione disulfide, and cysteine/cystine redox couples. These central couples, which we term redox control nodes, are maintained at stable but nonequilibrium steady states, are largely independently regulated in different subcellular compartments, and are quasi-independent from each other within compartments. Disruption of the redox control nodes can differentially affect sulfur switches, thereby creating a diversity of oxidative stress responses. Systems biology provides approaches to address the complexity of these responses. In the present review, we summarize thiol/disulfide pathway, redox potential, and rate information as a basis for kinetic modeling of sulfur switches. The summary identifies gaps in knowledge especially related to redox communication between compartments, definition of redox pathways, and discrimination between types of sulfur switches. A formulation for kinetic modeling of GSH/GSSG redox control indicates that systems biology could encourage novel therapeutic approaches to protect against oxidative stress by identifying specific redox-sensitive sites which could be targeted for intervention.     

Redox regulation of MAP kinase phosphatase 3

MAP kinase phosphatase 3 (MKP3) is a protein tyrosine phosphatase (PTP) for which in vivo evidence suggests that regulation can occur by oxidation and/or reduction of the active site cysteine. Using kinetics and mass spectrometry, we have probed the biochemical details of oxidation of the active site cysteine in MKP3, with particular focus on the mechanism of protection from irreversible inactivation to the sulfinic or sulfonic acid species. Like other PTPs, MKP3 was found to be rapidly and reversibly inactivated by mild treatment with hydrogen peroxide. We demonstrate that unlike the case for some PTPs, the sulfenic acid of the active site cysteine in MKP3 is not stabilized in the active site but instead is rapidly trapped in a re-reducible form. Unlike the case for other PTPs, the sulfenic acid in MKP3 does not form a sulfenyl-amide species with its neighboring residue or a disulfide with a single proximate cysteine. Instead, multiple cysteines distributed in both the N-terminal substrate-binding domain (Cys147 in particular) and the C-terminal catalytic domain (Cys218) are capable of rapidly and efficiently trapping the sulfenic acid as a disulfide. Our results extend the diversity of mechanisms utilized by PTPs to prevent irreversible oxidation of their active sites and expand the role of the N-terminal substrate recognition domain in MKP3 to include redox regulation.     

A chemical approach for detecting sulfenic acid-modified proteins in living cells

Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO2H) and sulfonic acids (Cys-SO3H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acid-modified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting protein sulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteine residues.     

Principles of redox control in photosynthesis gene expression

Light is one of the most important environmental factors influencing gene expression in photosynthetic organisms. In particular, genes for components of the photosynthetic machinery show light-dependent expression. In recent years, it has become clear that photosynthesis itself contributes important signals to this light control of gene expression by means of changes in the reduction/oxidation (redox) state of signalling molecules. Such changes in redox state are induced by changes in quality and quantity of the incident light. Redox signalling mechanisms therefore provide photosynthesis with the possibility of acclimational changes in the structure of the photosynthetic apparatus via a feedback control of photosynthesis gene expression. The great variety of these signalling mechanisms is summarised under the term 'redox control'. In some cases, oxygen acts as a different environmental, light-independent stimulus of photosynthetic gene expression, providing an additional redox signal and a different kind of redox control. In this review, we summarise present knowledge about such redox control mechanisms and analyse common properties as well as differences in the various signalling pathways. We suggest that there is an urgent need for a clear distinction between different kinds of redox control. Accordingly, we propose a categorisation into perceptional and transductional redox control. These categories are defined and examples given. The generalisation and comparability of results obtained in different physiological test systems and species are critically discussed.     

Reactive sulfur species: an emerging concept in oxidative stress

The ingredients of oxidative stress include a variety of reactive species such as reactive oxygen and reactive nitrogen species (ROS, RNS). While sulfur is usually considered as part of cellular antioxidant systems there is mounting evidence that reactive sulfur species (RSS) with stressor properties similar to the ones found in ROS are formed under conditions of oxidative stress. Thiols as well as disulfides are easily oxidised to sulfur species with sulfur in higher oxidation states. Such agents include thiyl radicals, disulfides, sulfenic acids and disulfide-S-oxides. They rapidly oxidise and subsequently inhibit thiol-proteins and enzymes and can be considered as a separate class of oxidative stressors providing new antioxidant drug targets.     

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.     

Thioredoxin-dependent redox regulation of cellular signaling and stress response through reversible oxidation of methionines

The sensitive oxidations of sulfur containing amino acids (i.e., cysteines and methionines) commonly control protein function, and act as important signaling mechanisms to modify metabolic responses to environmental stressors. Mechanisms associated with cysteine oxidation to form sulfenic acid and disulfides (i.e., cystine and glutathione adducts), and their reversibility through thioredoxin-dependent mechanisms, are broadly appreciated as important regulatory mechanisms that control the function of a range of different proteins. Less commonly understood are the cellular consequences of methionine oxidation to form methionine sulfoxide, as the structural requirements for their thioredoxin-dependent reduction by methionine sulfoxide reductases limit the reversibility of methionine oxidation to sequences within surface exposed and conformationally disordered regions of proteins. Surface exposed methionines are commonly involved in molecular recognition between transient protein signaling complexes, where their oxidation disrupts productive protein-protein interactions linked to a range of cellular responses. Such a signaling protein is calmodulin, which represents an early and central point in calcium signaling pathways important to stress responses in plants. We describe recent work elucidating fundamental mechanisms of reversible methionine oxidation within calmodulin, including the physical basis for differences in the sensitivity of individual methionines within plant and animal calmodulin to reactive oxygen species (ROS), the structural and functional consequences of their oxidation, and the interactions of oxidized calmodulin with methionine sulfoxide reductase enzymes. It is suggested that, in combination with high-throughput proteomic methods and current generation informatics tools, these mechanistic insights permit useful predictions of oxidatively sensitive signaling proteins that act as redox and stress sensors in response to methionine oxidation.     

Facile synthesis and biological evaluation of a cell-permeable probe to detect redox-regulated proteins

We have developed an improved synthesis for the cell-permeable, sulfenic acid probe DAz-1. Using DAz-1, we detect sulfenic acid modifications in the cell-cycle regulatory phosphatase Cdc25A. In addition, we show that DAz-1 has superior potency in cells compared to a biotinylated derivative. Collectively, these findings set the stage for the development of activity-based inhibitors of Cdc25 cell-cycle phosphatases, which are sensitive to the redox state of the active-site cysteine and demonstrate the advantage of bioorthogonal conjugation methods to detect protein sulfenic acids in cells.     

The chemistry behind redox regulation with a focus on sulphur redox systems

Sulphur metabolism in plants provides a wealth of natural products, including several chemically unusual substances, such as thiosulphinates, polysulphides and isothiocyanates. Many of these reactive sulphur species (RSS) exhibit a distinct redox behaviour in vitro, which translates into a rather interesting biological activity in vivo, such as antibiotic, fungicidal, pesticidal or anticancer activity. While the molecular basis for such activity has long remained obscure, research into sulphur-based redox systems during the past 5–10 years has achieved a better knowledge of the in vitro properties of RSS and has led to an improved understanding of their impact on intracellular redox signalling and control pathways in living cells. It has become apparent that the redox chameleon sulphur occurs in biological systems in about 10 different oxidation states, which give rise to an extensive and complicated network of sulphur-based redox events. Together, natural sulphur products from plants and their intracellular targets provide the basis for innovative design of novel antibiotics, fungicides, pesticides and anticancer agents.     

Generation of a cysteine sulfinic acid analog for incorporation in peptides using solid phase peptide synthesis

The sulfinic acid analog of aspartic acid, cysteine sulfinic acid, introduces a sulfur atom that perturbs the acidity and oxidation properties of aspartic acid. Cysteine sulfinic acids are often introduced in peptides and proteins by oxidation of cysteine, but this method is limited as all cysteine residues are oxidized and cysteine residues are often oxidized to sulfonic acids. To provide the foundation for the specific incorporation of cysteine sulfinic acids in peptides and proteins, we synthesized a 9-fluorenylmethyloxycarbonyl (Fmoc) benzothiazole sulfone analog. Oxidation conditions to generate the sulfone were examined and oxidation of the Fmoc-protected sulfide (3) with NbC in hydrogen peroxide provided the corresponding sulfone (4) in the highest yield and purity. Reduction with sodium borohydride generated the cysteine sulfinic acid (5) suggesting this approach may be an efficient method to incorporate a cysteine sulfinic acid in biomolecules. A model tripeptide bearing a cysteine sulfinic acid was synthesized using this approach. Future studies are aimed at using this method to incorporate cysteine sulfinic acids in peptide hormones and proteins for use in the study of biological function.     

Peptergents: peptide detergents that improve stability and functionality of a membrane protein, glycerol-3-phosphate dehydrogenase

Toward enhancing in vitro membrane protein studies, we have utilized small self-assembling peptides with detergent properties ("peptergents") to extract and stabilize the integral membrane flavoenzyme, glycerol-3-phosphate dehydrogenase (GlpD), and the soluble redox flavoenzyme, NADH peroxidase (Npx). GlpD is a six transmembrane spanning redox enzyme that catalyzes the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate. Although detergents such as n-octyl-beta-D-glucpyranoside can efficiently solubilize the enzyme, GlpD is inactivated within days once reconstituted into detergent micelles. In contrast, peptergents can efficiently extract and solubilize GlpD from native Escherichia coli membrane and maintain its enzymatic activity up to 10 times longer than in traditional detergents. Intriguingly, peptergents also extended the activity of a soluble flavoenzyme, Npx, when used as an additive. Npx is a flavoenzyme that catalyzes the two-electron reduction of hydrogen peroxide to water using a cysteine-sulfenic acid as a secondary redox center. The lability of the peroxidase results from oxidation of the sulfenic acid to the sulfinic or sulfonic acid forms. Oxidation of the sulfenic acid, the secondary redox center, results in inactivation, and this reaction proceeds in vitro even in the presence of reducing agents. Although the exact mechanism by which peptergents influence solution stability of Npx remains to be determined, the positive effects may be due to antioxidant properties of the peptides. Peptide-based detergents can be beneficial for many applications and may be particularly useful for structural and functional studies of membrane proteins due to their propensity to enhance the formation of ordered supramolecular assemblies.