RESUMO
Sulfenic acids are important intermediates in the oxidation of cysteine thiol groups in proteins by reactive oxygen species. The mechanism is influenced heavily by the presence of polar groups, other thiol groups, and solvent, all of which determines the need to compute precisely the energies involved in the process. Surprisingly, very scarce experimental information exists about a very basic property of sulfenic acids, the enthalpies of formation. In this Article, we use high level quantum chemical methods to derive the enthalpy of formation at 298.15 K of methane-, ethene-, ethyne-, and benzenesulfenic acids, the only ones for which some experimental information exists. The methods employed were tested against well-known experimental data of related species and extensive CCSD(T) calculations. Our best results consistently point out to a much lower enthalpy of formation of methanesulfenic acid, CH3SOH (ΔfH0(298.15K) = -35.1 ± 0.4 kcal mol-1), than the one reported in the NIST thermochemical data tables. The enthalpies of formation derived for ethynesulfenic acid, HC≡CSOH, +32.9 ± 1.0 kcal/mol, and benzenesulfenic acid, C6H5SOH, -2.6 ± 0.6 kcal mol-1, also differ markedly from the experimental values, while the enthalpy of formation of ethenesulfenic acid CH2CHSOH, not available experimentally, was calculated as -11.2 ± 0.7 kcal mol-1.
Assuntos
Cisteína , Ácidos Sulfênicos , Cisteína/química , Proteínas , Ácidos Sulfênicos/metabolismo , Compostos de Sulfidrila/química , TermodinâmicaRESUMO
Protein phosphorylation is a major post-translational modification involved in cell signalling that regulates many physiological and pathological processes. Despite their biological importance, protein phosphatases are less studied than protein kinases. Importantly, the activity of Cys-based protein tyrosine phosphatases (PTPs) can be regulated by reversible oxidation. The initial two-electron oxidation product of the active site Cys is a sulfenic acid (Cys-SOH) that can then undergo distinct outcomes, such as the disulfide bond or a sulfenyl amide formation. Here, we review the biochemical and structural features of PTPs to find patterns that might specify their oxidation products, aiming to get insights into redox regulatory mechanisms. Initially, the structure and biochemistry of PTP1B is presented. Then, we describe structural aspects that are relevant for substrate recognition and catalysis. Notably, all PTPs contain critical Cys residues for the catalysis of dephosphorylation that is prone to oxidative inactivation, which are frequently found oxidized in cells under physiological conditions, such as upon growth factor stimuli. However, direct oxidations of Cys residues in PTPs by H2 O2 are rather slow. Therefore, we discuss possible mechanisms that may account for this apparent contradiction between biological and chemical redox aspects of PTPs. Furthermore, we performed a systematic analysis of the distance between active site cysteine and its backdoor cysteine with the attempt to analyse the preference between disulfide bond formation or sulfenyl amide interaction upon oxidation. In summary, PTPs have been showing many possibilities to auto-protect from irreversible oxidation, which is important for cell signalling regulation.
Assuntos
Cisteína , Ácidos Sulfênicos , Amidas/química , Cisteína/química , Dissulfetos/metabolismo , Oxirredução , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismo , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Ácidos Sulfênicos/química , Ácidos Sulfênicos/metabolismoRESUMO
Sulfenic acids are the primary product of thiol oxidation by hydrogen peroxide and other oxidants. Several aspects of sulfenic acid formation through thiol oxidation were established recently. In contrast, the reduction of sulfenic acids is still scarcely investigated. Here, we characterized the kinetics of the reduction of sulfenic acids by ascorbate in several proteins. Initially, we described the crystal structure of our model protein (Tsa2-C170S). There are other Tsa2 structures in distinct redox states in public databases and all of them are decamers, with the peroxidatic cysteine very accessible to reductants, convenient features to investigate kinetics. We determined that the reaction between Tsa2-C170S-Cys-SOH and ascorbate proceeded with a rate constant of 1.40 ± 0.08 × 103 M-1 s-1 through a competition assay developed here, employing 2,6-dichlorophenol-indophenol (DCPIP). A series of peroxiredoxin enzymes (Prx6 sub family) were also analyzed by this competition assay and we observed that the reduction of sulfenic acids by ascorbate was in the 0.4-2.2 × 103 M-1 s-1 range. We also evaluated the same reaction on glyceraldehyde 3-phosphate dehydrogenase and papain, as the reduction of their sulfenic acids by ascorbate were reported previously. Once again, the rate constants are in the 0.4-2.2 × 103 M-1 s-1 range. We also analyzed the reduction of Tsa2-C170S-SOH by ascorbate by a second, independent method, following hydrogen peroxide reduction through a specific electrode (ISO-HPO-2, World Precision Instruments) and employing a bi-substrate, steady state approach. The kcat/KMAsc was 7.4 ± 0.07 × 103 M-1 s-1, which was in the same order of magnitude as the value obtained by the DCPIP competition assay. In conclusion, our data indicates that reduction of sulfenic acid in various proteins proceed at moderate rate and probably this reaction is more relevant in biological systems where ascorbate concentrations are high.
Assuntos
Ácidos Sulfênicos , Compostos de Sulfidrila , Cisteína/metabolismo , Peróxido de Hidrogênio , Oxirredução , Peroxirredoxinas/metabolismoRESUMO
Hydrogen sulfide (H2S) participates in prokaryotic metabolism and is associated with several physiological functions in mammals. H2S reacts with oxidized thiol derivatives (i.e. disulfides and sulfenic acids) and thereby forms persulfides, which are plausible transducers of the H2S-mediated signaling effects. The one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mycobacterium tuberculosis (MtAhpE-SH) reacts fast with hydroperoxides, forming a stable sulfenic acid (MtAhpE-SOH), which we chose here as a model to study the interactions between H2S and peroxiredoxins (Prx). MtAhpE-SOH reacted with H2S, forming a persulfide (MtAhpE-SSH) detectable by mass spectrometry. The rate constant for this reaction was (1.4 ± 0.2) × 103 m-1 s-1 (pH 7.4, 25 °C), six times higher than that reported for the reaction with the main low-molecular-weight thiol in M. tuberculosis, mycothiol. H2S was able to complete the catalytic cycle of MtAhpE and, according to kinetic considerations, it could represent an alternative substrate in M. tuberculosis. MtAhpE-SSH reacted 43 times faster than did MtAhpE-SH with the unspecific electrophile 4,4'-dithiodipyridine, a disulfide that exhibits no preferential reactivity with peroxidatic cysteines, but MtAhpE-SSH was less reactive toward specific Prx substrates such as hydrogen peroxide and peroxynitrite. According to molecular dynamics simulations, this loss of specific reactivity could be explained by alterations in the MtAhpE active site. MtAhpE-SSH could transfer its sulfane sulfur to a low-molecular-weight thiol, a process likely facilitated by the low pKa of the leaving thiol MtAhpE-SH, highlighting the possibility that Prx participates in transpersulfidation. The findings of our study contribute to the understanding of persulfide formation and reactivity.
Assuntos
Cisteína/análogos & derivados , Dissulfetos/metabolismo , Mycobacterium tuberculosis/metabolismo , Peroxirredoxinas/metabolismo , Catálise , Domínio Catalítico , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Peróxido de Hidrogênio/química , Sulfeto de Hidrogênio/metabolismo , Cinética , Oxirredução , Especificidade por Substrato , Ácidos Sulfênicos/metabolismo , Compostos de Sulfidrila/química , SulfetosRESUMO
Peroxiredoxins are thiol-dependent peroxidases that function in peroxide detoxification and H2 O2 induced signaling. Among the six isoforms expressed in humans, PRDX1 and PRDX2 share 97% sequence similarity, 77% sequence identity including the active site, subcellular localization (cytosolic) but they hold different biological functions albeit associated with their peroxidase activity. Using recombinant human PRDX1 and PRDX2, the kinetics of oxidation and hyperoxidation with H2 O2 and peroxynitrite were followed by intrinsic fluorescence. At pH 7.4, the peroxidatic cysteine of both isoforms reacts nearly tenfold faster with H2 O2 than with peroxynitrite, and both reactions are orders of magnitude faster than with most protein thiols. For both isoforms, the sulfenic acids formed are in turn oxidized by H2 O2 with rate constants of ca 2 × 103 M-1 s-1 and by peroxynitrous acid significantly faster. As previously observed, a crucial difference between PRDX1 and PRDX2 is on the resolution step of the catalytic cycle, the rate of disulfide formation (11 s-1 for PRDX1, 0.2 s-1 for PRDX2, independent of the oxidant) which correlates with their different sensitivity to hyperoxidation. This kinetic pause opens different pathways on redox signaling for these isoforms. The longer lifetime of PRDX2 sulfenic acid allows it to react with other protein thiols to translate the signal via an intermediate mixed disulfide (involving its peroxidatic cysteine), whereas PRDX1 continues the cycle forming disulfide involving its resolving cysteine to function as a redox relay. In addition, the presence of C83 on PRDX1 imparts a difference on peroxidase activity upon peroxynitrite exposure that needs further study.
Assuntos
Peróxido de Hidrogênio/química , Peroxirredoxinas/química , Ácido Peroxinitroso/química , Humanos , Cinética , Oxirredução , Proteínas Recombinantes/química , Ácidos Sulfênicos/químicaRESUMO
Hydrogen sulfide (H2S) has been traditionally considered to be a toxic molecule for mammals. However, it can be formed endogenously and exert physiological effects with potential health benefits. H2S can partition two-fold in biological membranes and traverse them rapidly, diffusing between compartments. H2S reactivity has similarities to that of thiols, although it is less nucleophilic than thiols and it can form different products. H2S can react with oxidants derived from the partial reduction of oxygen, but direct scavenging is unlikely to explain H2S protective actions. Important effects are exerted on mitochondria including the stimulation or the inhibition of the electron transport chain. Possible mechanisms for unleashing biological consequences are the reactions with metal centers and with thiol oxidation products. The reactions of H2S with disulfides (RSSR) and sulfenic acids (RSOH) lead to the formation of persulfides (RSSH). Persulfides have enhanced nucleophilicity with respect to the corresponding thiol, consistent with the alpha effect. Besides, the inner and outer sulfurs can both act as electrophiles. In this review, we describe the reactions of H2S with oxidized thiol products and the properties of the persulfides formed in the context of the chemical biology of H2S.
Assuntos
Sulfeto de Hidrogênio/química , Sulfetos/química , Dissulfetos/química , Elétrons , Gases , Metais/química , Mitocôndrias/metabolismo , Oxigênio/química , Permeabilidade , Transdução de Sinais , Ácidos Sulfênicos/química , Compostos de Sulfidrila/química , Enxofre/químicaRESUMO
The oxidation of biothiols participates not only in the defense against oxidative damage but also in enzymatic catalytic mechanisms and signal transduction processes. Thiols are versatile reductants that react with oxidizing species by one- and two-electron mechanisms, leading to thiyl radicals and sulfenic acids, respectively. These intermediates, depending on the conditions, participate in further reactions that converge on different stable products. Through this review, we will describe the biologically relevant species that are able to perform these oxidations and we will analyze the mechanisms and kinetics of the one- and two-electron reactions. The processes undergone by typical low-molecular-weight thiols as well as the particularities of specific thiol proteins will be described, including the molecular determinants proposed to account for the extraordinary reactivities of peroxidatic thiols. Finally, the main fates of the thiyl radical and sulfenic acid intermediates will be summarized.
Assuntos
Compostos de Sulfidrila/metabolismo , Animais , Bactérias , Eucariotos , Radicais Livres , Humanos , Cinética , Oxirredução , Ácidos Sulfênicos , Compostos de Sulfidrila/químicaRESUMO
Hydrogen sulfide (H2S) is increasingly recognized to modulate physiological processes in mammals through mechanisms that are currently under scrutiny. H2S is not able to react with reduced thiols (RSH). However, H2S, more precisely HS(-), is able to react with oxidized thiol derivatives. We performed a systematic study of the reactivity of HS(-) toward symmetric low molecular weight disulfides (RSSR) and mixed albumin (HSA) disulfides. Correlations with thiol acidity and computational modeling showed that the reaction occurs through a concerted mechanism. Comparison with analogous reactions of thiolates indicated that the intrinsic reactivity of HS(-) is 1 order of magnitude lower than that of thiolates. In addition, H2S is able to react with sulfenic acids (RSOH). The rate constant of the reaction of H2S with the sulfenic acid formed in HSA was determined. Both reactions of H2S with disulfides and sulfenic acids yield persulfides (RSSH), recently identified post-translational modifications. The formation of this derivative in HSA was determined, and the rate constants of its reactions with a reporter disulfide and with peroxynitrite revealed that persulfides are better nucleophiles than thiols, which is consistent with the α effect. Experiments with cells in culture showed that treatment with hydrogen peroxide enhanced the formation of persulfides. Biological implications are discussed. Our results give light on the mechanisms of persulfide formation and provide quantitative evidence for the high nucleophilicity of these novel derivatives, setting the stage for understanding the contribution of the reactions of H2S with oxidized thiol derivatives to H2S effector processes.
Assuntos
Dissulfetos/metabolismo , Sulfeto de Hidrogênio/metabolismo , Ácidos Sulfênicos/metabolismo , Sulfetos/metabolismo , Linhagem Celular , Dissulfetos/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Sulfeto de Hidrogênio/química , Técnicas In Vitro , Cinética , Modelos Biológicos , Modelos Químicos , Peso Molecular , Oxirredução , Albumina Sérica/química , Albumina Sérica/metabolismo , Ácidos Sulfênicos/química , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Sulfetos/químicaRESUMO
Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function.
Assuntos
Amidas/química , Cisteína/química , Proteínas/química , Proteínas/metabolismo , Ácidos Sulfênicos/química , Amidas/metabolismo , Biologia Computacional , Cisteína/metabolismo , Modelos Moleculares , Oxirredução , Conformação Proteica , Ácidos Sulfênicos/metabolismoRESUMO
Evidence has accumulated showing that hydrogen peroxide (H2O2) acts as a signaling molecule via oxidation of critical cysteine residues on target proteins. The reaction of H2O2 with thiols is thermodynamically favored, but its selectivity is imposed by differences in reaction kinetics. Previously proposed signal relaying mechanisms, such as the floodgate hypothesis and widespread protein sulfenylation, appear inconsistent with kinetic and diffusion considerations. Among all cellular thiols, the peroxidatic cysteines of peroxiredoxins (Prxs) represent preferential targets considering their high rate constants and their cellular abundance that place them as the first step in the H2O2-induced signaling pathways. The oxidized Prxs could transfer the signal either via thiol-disulfide redox reactions or through nonredox protein-protein interactions. Recent studies evidence Prxs interactions with protein tyrosine kinases and phosphatases, indicating a potential connection between redox and phosphorylation signaling pathways that does not need the direct reaction of H2O2 with phosphatase or kinase critical cysteines. Posttranslational modifications of Prxs have been observed in vivo (mainly overoxidation of cysteines and phosphorylation of threonines) that affect their peroxidase activity, redox state, and/or oligomeric structure, and likely impact on H2O2 signaling. More focus on kinetic data and redox-sensitive protein-protein interactions are needed to unravel the molecular mechanisms of H2O2 signaling.