Isolable small-molecule cysteine sulfenic acid.

B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 min. Increased ROIs preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration-dependent block in anti-IgM-induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.

Nitrile Hydratase (NHase) from Rhodococcus sp. N771 contains a mononuclear non-heme iron center. NHase is inactivated by nitrosylation. The nitric monoxide molecule on the iron center is dissociated by photo-irradiation, possibly replaced with a water molecule in the active state. Sulfur atoms of Cys112 and Cys114, coordinating to the iron center, are post-translationally modified to cysteine-sulfinic (Cys112-SO2H) and cysteine-sulfenic acid (Cys114-SOH), respectively (Nat. Struct. Biol., 5, 347-351, 1998). To study the photoactivation process and the reaction mechanism of NHase, we prepared the crystals of the complex between inactive NHase and an irreversible inhibitor, cyclohexyl isocyanide by soaking experiments and investigated the structural changes induced by photo-irradiation. The crystals were irradiated with a normal fluorescent lamp (14 mW/cm 2 ) for 0hr, 0.75hr, 1.0hr, 1.5hr, and 2.0hr at 4C and stored in the liquid nitrogen. X-ray diffraction data sets for the crystals were collected in the light using BL44B2 at SPring-8. Crystal structures were refined using CNS and SHELX-97 at 1.5, 1.6, 1.7, 1.2, and 1.7 resolutions to R-factors of 15.5, 14.9, 15.5, 15.5, and 14.8%, respectively. Upon photoactivation, the isocyanide group of the inhibitor turned to the opposite direction and bound directly to the iron. Surprisingly, the electron density for the O of Cys114-SOH gradually decreased and disappeared at 1.0hr photo-irradiation, but appeared again in the crystals further irradiated. O of Cys114-SOH might be replaced with an oxygen atom of water molecule during inhibitor binding to NHase.

Oxidative transformation of cysteine thiol groups into different functional groups is considered a significant posttranslational modification of great importance in pathological and physiological processes. A cysteine sulfenic acid (SA) residue is the transient state for thiol group oxidation and it can react with free thiols to form disulfide bonds or can be further oxidized with reactive oxygen/reactive nitrogen species (ROS/RNS) to form sulfinic and sulfonic acids. The increase in ROS/RNS concentrations is correlated to age-related diseases such as cancer and Alzheimer's disease. Since the formation of SA represents a transient state of oxidation of thiols, its formation can be considered a redox-sensitive sensor for the presence of ROS/RNS. Thereby, the detection of the short-lived SA will provide greater insight into the redox-mediated events that alter the structure and function of peptides and proteins. The aim of this study is to provide a new strategy for the highly sensitive and specific detection of SA in peptides as a proof of concept. For this aim, SA was firstly generated in model peptides on oxidation with H2O2 and then captured by the linear alkyne β-ketoester (KE) previously linked to a lanthanide (Ln)-containing chelator (Ln-DOTA, where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The linking of the KE to DOTA was performed by click chemistry, resulting in a new reagent (Ln-DOTA-KE) that permits highly sensitive elemental (inductively coupled plasma) and molecular (electrospray) mass spectrometric detection. The new reagent (Ln-DOTA-KE) reacts specifically with SA, offering improved reactivity at physiological pH, facile derivatization and a cell-membrane-permeable compound that has promising future applications. Graphical Abstract A new derivatizing reagent for specific detection of sulphenic acid (SA) generated in model peptides by oxidation of cysteine groups is presented in this work.

Cysteine sulfenyl iodides (Cys–SIs) have long been recognized as important intermediates in the oxidative modification of cysteine thiols since 1950s, implicated in pathways such as hydrolysis to cysteine sulfenic acid (Cys–SOH) and electrophilic aromatic substitution with an indole ring of tryptophan to form Cys–Trp thioether linkages. Despite their proposed significance in both biological and chemical contexts, direct examination of Cys–SIs has been precluded by their intrinsic instability. Herein, the first isolation of a small‐molecule Cys–SI, stabilized using a nanosized molecular cradle at the N‐terminus of cysteine, is reported. The structure of the isolable Cys–SI is determined by X‐ray crystallography, and its reactivity is investigated with a range of nucleophiles. Hydrolysis to Cys–SOH and reaction with an indole derivative provide direct chemical evidence for long‐standing mechanistic proposals. Furthermore, rapid formation of sulfenamides with amines and high‐yield adduct formation with dimedone, a canonical sulfenic‐acid probe, reveal that Cys–SI possesses even greater electrophilicity than Cys–SOH. These results deliver a structurally defined reference for Cys–SI and inform mechanisms of iodine‐mediated protein oxidation and peptide modification.

<p>Cysteine sulfenic acids are thought to be key intermediates in protein oxidation but are difficult to study due to their high reactivity and transient nature. The beneficial properties of these oxidative events have been underexplored due to earlier studies primarily focusing on cell damage and death; however, increasing evidence suggests various physiological processes rely on these oxidative modifications. Previous work has shown that bicyclo[6.1.0]non-4-ynes (BCNs) can label sulfenic acids with high reaction kinetics via a pericyclic reaction, while also enabling copper-free click chemistry, allowing for a biotin-azide tag to be installed. However, due to the strong biotin- streptavidin interaction, these probes suffer from poor protein recovery and sub-optimal performance during mass-spectrometric analysis, respectively. Efforts towards incorporating the BCNs into a novel symmetrical photocleavable group based on 1,4-naphthoquinone, or their use in combination with photocleavable biotin-azide are proposed to improve upon these limitations and are presented in this thesis. </p>

Reactive oxygen species act as important second messengers in cell signaling and homeostasis through the oxidation of protein thiols. However, the dynamic nature of protein oxidation and the lack of sensitivity of existing molecular probes have hindered our understanding of such reactions; therefore, new tools are required to address these challenges. We designed a bifunctional variant of the strained bicyclo[6.1.0]nonyne (BCN-E-BCN) that enables the tagging of intracellular protein sulfenic acids for biorthogonal copper-free click chemistry. In validation studies, BCN-E-BCN binds the sulfenylated form of the actin-severing protein cofilin, while mutation of the cognate cysteine residues abrogates its binding. BCN-E-BCN is cell permeable and reacts rapidly with cysteine sulfenic acids in cultured cells. Using different azide-tagged conjugates, we demonstrate that BCN-E-BCN can be used in various applications for the detection of sulfenylated proteins. Remarkably, cycloaddition of an azide-tagged fluorophore to BCN-E-BCN labeled proteins produced in vivo can be visualized by fluorescence microscopy to reveal their localization. These findings demonstrate a novel and multifaceted approach to the detection and trapping of sulfenic acids.

The Atomic Resolution Crystal Structure of the YajL (ThiJ) Protein from Escherichia coli: A Close Prokaryotic Homologue of the Parkinsonism-associated Protein DJ-1

Nitrile hydratase (NHase) is one of a growing number of enzymes shown to contain post-translationally modified cysteine sulfenic acids (Cys-SOH). Cysteine sulfenic acids have been shown to play diverse roles in cellular processes, including transcriptional regulation, signal transduction, and the regulation of oxygen metabolism and oxidative stress responses. The function of the cysteine sulfenic acid coordinated to the iron active site of NHase is unknown. Herein we report the first example of a sulfenate-ligated iron complex, [Fe(III)(ADIT)(ADIT-O)](+) (5), and compare its electronic and magnetic properties with those of structurally related complexes in which the sulfur oxidation state and protonation state have been systematically altered. Oxygen atom addition was found to decrease the unmodified thiolate Fe-S bond length and blue-shift the ligand-to-metal charge-transfer band (without loss of intensity). S K-edge X-ray absorption spectroscopy and density functional theory calculations show that, although the modified RS-O(-) fragment is incapable of forming a pi bond with the Fe(III) center, the unmodified thiolate compensates for this loss of pi bonding by increasing its covalent bond strength. The redox potential shifts only slightly (75 mV), and the magnetic properties are not affected (the S = (1)/(2) spin state is maintained). The coordinated sulfenate S-O bond is activated and fairly polarized (S(+)-O(-)). Addition of strong acids at low temperatures results in the reversible protonation of sulfenate-ligated 5. An X-ray structure demonstrates that Zn(2+) binds to the sulfenate oxygen to afford [Fe(III)(ADIT)(ADIT-O-ZnCl(3))] (6). The coordination of ZnCl(3)(-) to the RS-O(-) unit causes the covalent overlap with the unmodified thiolate to increase further. A possible catalytic role for the unmodified NHase thiolate, involving its ability to "tune" the electronics in response to protonation of the sulfenate (RS-O(-)) oxygen and/or substrate binding, is discussed.

Remarkable stability and catalytic performance of PW11M@MOF-808 (M=Mn and Cu) nanocomposites for oxidative desulfurization of fuel oil

Peroxiredoxin 6 homodimerization and heterodimerization with glutathione S-transferase pi are required for its peroxidase but not phospholipase A2 activity

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.

Nitrile hydratases (NHase), which catalyze the hydration of nitriles to amides, have an unusual Fe(3+) or Co(3+) center with two modified Cys ligands: cysteine sulfininate (Cys-SO(2) (-)) and either cysteine sulfenic acid or cysteine sulfenate [Cys-SO(H)]. Two catalytic mechanisms have been proposed. One is that the sulfenyl oxygen activates a water molecule, enabling nucleophilic attack on the nitrile carbon. The other is that the Ser ligand ionizes the strictly conserved Tyr, activating a water molecule. Here, we characterized mutants of Fe-type NHase from Rhodococcus erythropolis N771, replacing the Ser and Tyr residues, alphaS113A and betaY72F. The alphaS113A mutation partially affected catalytic activity and did not change the pH profiles of the kinetic parameters. UV-vis absorption spectra indicated that the electronic state of the Fe center was altered by the alphaS113A mutation, but the changes could be prevented by a competitive inhibitor, n-butyric acid. The overall structure of the alphaS113A mutant was similar to that of the wild type, but significant changes were observed around the catalytic cavity. Like the UV-vis spectra, the changes were compensated by the substrate or product. The Ser ligand is important for the structure around the catalytic cavity, but is not essential for catalysis. The betaY72F mutant exhibited no activity. The structure of the betaY72F mutant was highly conserved but was found to be the inactivated state, with alphaCys114-SO(H) oxidized to Cys-SO(2) (-), suggesting that betaTyr72 affected the electronic state of the Fe center. The catalytic mechanism is discussed on the basis of the results obtained.

Synthesis of a rhodium(III) triphenylphosphine complex via C[sbnd]S bond cleavage of an azo-thioether ligand: X-ray structure, electrochemistry and catalysis towards transfer hydrogenation of ketones

Endosomal H2O2 production leads to localized cysteine sulfenic acid formation on proteins during lysophosphatidic acid-mediated cell signaling

Methionine sulfoxide reductase A (msrA) reduces methionine sulfoxide in proteins back to methionine. Its catalytic cysteine (Cys72-SH) has a low pKa that facilitates oxidation by methionine sulfoxide to cysteine sulfenic acid. If the catalytic cycle proceeds efficiently, the sulfenic acid is reduced back to cysteine at the expense of thioredoxin. However, the sulfenic acid is vulnerable to "irreversible" oxidation to cysteine sulfinic acid that inactivates msrA (hyperoxidation). We observed that human msrA is resistant to hyperoxidation while mouse msrA is readily hyperoxidized by micromolar concentrations of hydrogen peroxide. We investigated the basis of this difference in susceptibility to hyperoxidation and established that it is controlled by the presence or absence of a Met residue in the carboxyl-terminal domain of the enzyme, Met229. This residue is Val in human msrA, and when it was mutated to Met, human msrA became sensitive to hyperoxidation. Conversely, mouse msrA was rendered insensitive to hyperoxidation when Met229 was mutated to Val or one of five other residues. Positioning of the methionine at residue 229 is not critical, as hyperoxidation occurred as long as the methionine was located within the group of 14 carboxyl-terminal residues. The carboxyl domain of msrA is known to be flexible and to have access to the active site, and Met residues are known to form stable, noncovalent bonds with aromatic residues through interaction of the sulfur atom with the aromatic ring. We propose that Met229 forms such a bond with Trp74 at the active site, preventing formation of a protective sulfenylamide with Cys72 sulfenic acid. As a consequence, the sulfenic acid is available for facile, irreversible oxidation to cysteine sulfinic acid.