Cysteine-containing Proteins Research Articles

Orthogonal bioconjugation targeting cysteine-containing peptides and proteins using alkyl thianthrenium salts

Late-stage specific and selective diversifications of peptides and proteins performed at target residues under ambient conditions are recognized to be the most facile route to various and abundant conjugates. Herein, we report an orthogonal modification of cysteine residues using alkyl thianthreium salts, which proceeds with excellent chemoselectivity and compatibility under mild conditions, introducing a diverse array of functional structures. Crucially, multifaceted bioconjugation is achieved through clickable handles to incorporate structurally diverse functional molecules. This “two steps, one pot” bioconjugation method is successfully applied to label bovine serum albumin. Therefore, our technique is a versatile and powerful tool for late-stage orthogonal bioconjugation.

A Mass Spectrometry Strategy for Protein Quantification Based on the Differential Alkylation of Cysteines Using Iodoacetamide and Acrylamide.

Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.

Cysteine-Specific Multifaceted Bioconjugation of Peptides and Proteins Using 5-Substituted 1,2,3-Triazines.

Peptide and protein postmodification have gained significant attention due to their extensive impact on biomolecule engineering and drug discovery, of which cysteine-specific modification strategies are prominent due to their inherent nucleophilicity and low abundance. Herein, the study introduces a novel approach utilizing multifunctional 5-substituted 1,2,3-triazine derivatives to achieve multifaceted bioconjugation targeting cysteine-containing peptides and proteins. On the one hand, this represents an inaugural instance of employing 1,2,3-triazine in biomolecular-specific modification within a physiological solution. On the other hand, as a powerful combination of precision modification and biorthogonality, this strategy allows for the one-pot dual-orthogonal functionalization of biomolecules utilizing the aldehyde group generated simultaneously. 1,2,3-Triazine derivatives with diverse functional groups allow conjugation to peptides or proteins, while bi-triazines enable peptide cyclization and dimerization. The examination of the stability of bi-triazines revealed their potential for reversible peptide modification. This work establishes a comprehensive platform for identifying cysteine-selective modifications, providing new avenues for peptide-based drug development, protein bioconjugation, and chemical biology research.

Changes in the level of metallothioneins, zinc and copper in blood plasma of patients with chronic lymphocytic leukemia

The relationship between the content of metallothioneins, copper and zinc ions in peripheral blood plasma in chronic lymphocytic leukemia (CLL) depending on the disease progression was studied. It was shown that in patients with CLL in blood plasma with a progressive disease, partial and complete remission, the values of the zinc-to-copper ratios significantly decreased in comparison to similar indicators of practically healthy donors. It was established that in patients with CLL, the level of metallothioneins in blood plasma decreases with the disease progression compared to other studied groups, which indicates a reduced function of cysteine-containing metallothionein proteins. Based on the obtained data, it can be concluded that the zinc-to-copper concentration ratio and the level of metallothionenins in peripheral blood plasma are potential diagnostic and prognostic markers in chronic leukemia.

Oxidized and Reduced Dimeric Protein Complexes Illustrate Contrasting CID and SID Charge Partitioning.

Charge partitioning during the dissociation of protein complexes in the gas phase is influenced by many factors, such as interfacial interactions, protein flexibility, protein conformation, and dissociation methods. In the present work, two cysteine-containing homodimer proteins, β-lactoglobulin and α-lactalbumin, with the disulfide bonds intact and reduced, were used to gain insight into the charge partitioning behaviors of collision-induced dissociation (CID) and surface-induced dissociation (SID) processes. For these proteins, we find that restructuring dominates with CID and dissociation with symmetric charge partitioning dominates with SID, regardless of whether intramolecular disulfide bonds are oxidized or reduced. CID of the charge-reduced dimeric protein complex leads to a precursor with a slightly smaller collision cross section (CCS), greater stability, and more symmetrically distributed charges than the significantly expanded form produced by CID of the higher charged dimer. Collision-induced unfolding plots demonstrate that the unfolding-restructuring of the protein complexes initiates the charge migration of higher charge-state precursors. Overall, gas collisions reveal the charge-dependent restructuring/unfolding properties of the protein precursor, while surface collisions lead predominantly to more charge-symmetric monomer separation. CID's multiple low-energy collisions sequentially reorganize intra- and intermolecular bonds, while SID's large-step energy jump cleaves intermolecular interfacial bonds in preference to reorganizing intramolecular bonds. The activated population of precursors that have taken on energy without dissociating (populated in CID over a wide range of collision energies, populated in SID for only a narrow distribution of collision energies near the onset of dissociation) is expected to be restructured, regardless of the activation method.

Toxin and phage production from pathogenic E. coli by antibiotic induction analyzed by chemical reduction, MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis.

Shiga toxin-producing Escherichia coli (STEC) are an on-going threat to public health and agriculture. Our laboratory has developed a rapid method for identification of Shiga toxin (Stx), bacteriophage and host proteins produced from STEC. We demonstrate this technique on two genomically sequenced STEC O145:H28 strains linked to two major outbreaks of foodborne illness occurring in 2007 (Belgium) and 2010 (Arizona). Our approach is to induce expression of stx, prophage and host genes by antibiotic exposure, chemically reduce samples and identify protein biomarkers from unfractionated samples using MALDI-TOF-TOF, tandem mass spectrometry (MS/MS) and post-source decay (PSD). The protein mass and prominent fragment ions were used to identify protein sequences using top-down proteomic software developed in-house. Prominent fragment ions are the result of polypeptide backbone cleavage (PBC) resulting from the aspartic acid effect fragmentation mechanism. The B-subunit of Stx and acid-stress proteins HdeA and HdeB were identified in both STEC strains in their intramolecular disulfide bond-intact and reduced states. In addition, two cysteine-containing phage tail proteins were detected and identified from the Arizona strain but only under reducing conditions which suggests that bacteriophage complexes are bound by intermolecular disulfide bonds. An acyl carrier protein (ACP) and a phosphocarrier protein (HPr) were also identified from the Belgium strain. ACP was post-translationally modified with attachment of a phosphopantetheine linker at residue S36. The abundance of ACP (plus linker) was significantly increased upon chemical reduction suggesting the release of fatty acids bound to the ACP + linker at a thioester bond. MS/MS-PSD revealed dissociative loss of the linker from the precursor ion as well as fragment ions with and without the attached linker consistent with its attachment at S36. This study demonstrates the advantages of chemical reduction in facilitating the detection and top-down identification of protein biomarkers of pathogenic bacteria.

Cross-linking between cysteine and lysine, tryptophan or tyrosine in peptides and proteins treated with hypochlorous acid and other reactive halogens

Cysteine residues are the most favored targets for oxidation by hypochlorous acid and other reactive halogen species. The end-products of cysteine oxidation are usually considered to be reversibly formed disulfides and the more highly oxidized sulfinic and sulfonic acids. However, reactive halogen species are capable of generating additional products in which cysteine is cross-linked to other amino acids. Here we have treated a range of peptides with hypochlorous acid (HOCl) and hypobromous acid (HOBr), and used mass spectrometry to demonstrate sulfenamide, sulfinamide and sulfonamide formation with lysine residues, as well as –S(O)- and –S(O2)- linkages with tyrosine, tryptophan and arginine residues. The -(SO2)- products were more prevalent with HOCl than HOBr, reflecting its higher oxidizing ability. There was also considerable variation between peptides in efficiency of cross-linking compared with other modifications. The –S(O)- and –S(O2)- forms were much more resistant than the disulfide to reduction by dithiothreitol. Using calprotectin as a representative cysteine-containing protein, we show that a range of products containing each of these cross-links is formed when the protein is treated with HOCl. Two of the identified cysteine-lysine calprotectin cross-links were also detected in bronchoalveolar lavage fluid from children with cystic fibrosis. Our results imply that cross-linked species would be formed when cysteine-containing proteins are exposed to reactive halogen species, with the nature of the specific products depending on structural features around the cysteine residue. Cross-linking could have a modulatory effect on protein function or be detrimental in causing oligomerization and aggregation.

Thia-Michael addition: the route to promising opportunities for fast and cysteine-specific modification.

Over the last few decades, design and discovery of chemical reactions that enable modification of proteins at pre-determined sites have been the focus of synthetic organic chemists. As an invaluable tool, the site-and chemoselective functionalization of peptides and proteins offers an exciting opportunity for creating high-value multicomponent conjugates with diverse applications in life sciences and pharmacology. In recent years, multiple strategies have emerged that target natural amino acids directly or convert them into other reactive species for further ligations. However, reactivity and selectivity are still key issues in the current state of chemical modification methodologies. Cysteine is one of the least abundant amino acids and exhibits unique chemistry of the thiol or thiolate group which makes it susceptible to a series of post-translational modifications. The thia-Michael "click" addition reactions, which can proceed under facile conditions provide a promising way for thiol-selective modification of cysteine-containing proteins. In this review, we summarize various reactions for cysteine-selective peptide and protein modification, focus on thia-Michael "click" addition reactions, elaborate on their historical perspective and mechanism, and highlight their applications in modifying biomolecules in a site-specific way.

Carbonylacrylic Derivatives and Their Ability to Disrupt Biofi lm Formation

The rapid emergence of bacterial resistance to antibiotics has greatly pressed the need for novel therapeutics. One of the strategies bacteria use to enhance their resistance toward antibiotics is to embed themselves into polymeric matrices known as a biofi lm. Bacteria in a biofi lm state are highly resistant towards antibiotics and immune response, thereby more diffi cult to eradicate. Hence, antibiofi lm agents are considered an alternative strategy to unravel problems regarding bacterial antibiotic resistance. Our lab has focused on bacterial communication process called quorum sensing (QS), which is crucial for bacteria to conduct group behaviors such as biofi lm. Cysteine residue of LasR, key QS protein regulator, has been suggested to play an important role in QS-mediated biofi lm formation. In addition, cysteine-containing biomolecules have been implicated in bacterial pathogenesis. Herein, we explored the ability of thiol-reactive molecules for their ability to interfere with biofi lm formation in pathogenic Gram-negative Pseudomonas aeruginosa (P. aeruginosa). We hypothesized that the thiol-reactive molecules could potentially react with sulfhydryl group of cysteine residue of crucial QS enzyme or of cysteine-containing biomolecules that may lead to the reduce in the ability of the pathogen to form biofi lm. Carbonylacrylic compounds were previously reported to rapidly undergo thiol-Michael reaction with cysteine-containing proteins under physiological conditions. Total of three new carbonylacrylic derivatives were synthesized and tested for their anti-biofi lm activity. All synthesized compounds could inhibit biofi lm formation in a concentration-dependent manner without toxicity to bacteria. Derivative 1a exhibited the most potent antibiofi lm activity with IC50 of 85 μM.

Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents.

Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody–drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol (CPO-PFP), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2×cys-GFP, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the β-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.

Massive expression of cysteine-containing proteins causes abnormal elongation of yeast cells by perturbing the proteasome.

The enhanced green fluorescent protein (EGFP) is considered to be a harmless protein because the critical expression level that causes growth defects is higher than that of other proteins. Here, we found that overexpression of EGFP, but not a glycolytic protein Gpm1, triggered the cell elongation phenotype in the budding yeast Saccharomyces cerevisiae. By the morphological analysis of the cell overexpressing fluorescent protein and glycolytic enzyme variants, we revealed that cysteine content was associated with the cell elongation phenotype. The abnormal cell morphology triggered by overexpression of EGFP was also observed in the fission yeast Schizosaccharomyces pombe. Overexpression of cysteine-containing protein was toxic, especially at high-temperature, while the toxicity could be modulated by additional protein characteristics. Investigation of protein aggregate formation, morphological abnormalities in mutants, and transcriptomic changes that occur upon overexpression of EGFP variants suggested that perturbation of the proteasome by the exposed cysteine of the overexpressed protein causes cell elongation. Overexpression of proteins with relatively low folding properties, such as EGFP, was also found to promote the formation of SHOTA (Seventy kDa Heat shock protein-containing, Overexpression-Triggered Aggregates), an intracellular aggregate that incorporates Hsp70/Ssa1, which induces a heat shock response, while it was unrelated to cell elongation. Evolutionary analysis of duplicated genes showed that cysteine toxicity may be an evolutionary bias to exclude cysteine from highly expressed proteins. The overexpression of cysteine-less moxGFP, the least toxic protein revealed in this study, would be a good model system to understand the physiological state of protein burden triggered by ultimate overexpression of harmless proteins.

Antibacterial activity evaluation of vinyl sulfones against global predominant methicillin-resistant Staphylococcus aureus USA300

The electrophilic potential of vinyl sulfone permits the rapid capture of cysteine-containing proteins under physiological conditions. These cysteine proteinases play vital roles in bacterial survival and pathogenesis of Staphylococcus aureus (S. aureus) and the global health threat methicillin resistant S. aureus (MRSA). Here in, total of 28 vinyl sulfones were synthesized and subjected to susceptibility testing of pathogenic bacteria, including global epidemic MRSA PFGE strain type USA300 (SF8300). Number of antibacterial vinyl sulfone derivatives were discovered. Among these, nitrile-substituted vinyl phenyl sulfones showed potent antibacterial activity. (E)-3-((4-methoxyphenyl)sulfonyl)acrylonitrile exhibited the strongest potency with MIC of 1.875 µg/mL against methicillin susceptible S. aureus and 3.75 µg/mL against MRSA USA300. Based on the structure–activity relationship analysis, the antibacterial activity of these compounds may involve sulfhydryl conjugation. In addition, the nitrile-substituted vinyl phenyl sulfone could also impair host cell adhesion. With their promising antibacterial activities, these vinyl sulfones have potential for S. aureus and MRSA therapeutics.

Functionalized quinolizinium-based fluorescent reagents for modification of cysteine-containing peptides and proteins.

A series of quinolizinium-based fluorescent reagents were prepared by visible light-mediated gold-catalyzed cis-difunctionalization between quinolinium diazonium salts and electron-deficient alkyne-linked phenylethynyl trimethylsilanes. The electron-deficient alkynyl group of the quinolizinium-based fluorescent reagents underwent nucleophilic addition reaction with the sulfhydryl group on cysteine-containing peptides and proteins. The quinolizinium-based fluorescent reagents were found to function as highly selective reagents for the modification of cysteine-containing peptides and proteins with good to excellent conversions (up to 99%). Moreover, the modified BCArg mutants bearing cationic quinolizinium compounds 1b, 1d, 1e and 1h exhibit comparable activity in enzymatic and cytotoxicity assays to the unmodified one.

Crystal structure of human cysteamine dioxygenase provides a structural rationale for its function as an oxygen sensor

Cysteamine dioxygenase (ADO) plays a vital role in regulating thiol metabolism and preserving oxygen homeostasis in humans by oxidizing the sulfur of cysteamine and N-terminal cysteine-containing proteins to their corresponding sulfinic acids using O2 as a cosubstrate. However, as the only thiol dioxygenase that processes both small-molecule and protein substrates, how ADO handles diverse substrates of disparate sizes to achieve various reactions is not understood. The knowledge gap is mainly due to the three-dimensional structure not being solved, as ADO cannot be directly compared with other known thiol dioxygenases. Herein, we report the first crystal structure of human ADO at a resolution of 1.78 Å with a nickel-bound metal center. Crystallization was achieved through both metal substitution and C18S/C239S double mutations. The metal center resides in a tunnel close to an entry site flanked by loops. While ADO appears to use extensive flexibility to handle substrates of different sizes, it also employs proline and proline pairs to maintain the core protein structure and to retain the residues critical for catalysis in place. This feature distinguishes ADO from thiol dioxygenases that only oxidize small-molecule substrates, possibly explaining its divergent substrate specificity. Our findings also elucidate the structural basis for ADO functioning as an oxygen sensor by modifying N-degron substrates to transduce responses to hypoxia. Thus, this work fills a gap in structure–function relationships of the thiol dioxygenase family and provides a platform for further mechanistic investigation and therapeutic intervention targeting impaired oxygen sensing.

Cysteine Proteome Reveals Response to Endogenous Oxidative Stress in Bacillus cereus.

At the end of exponential growth, aerobic bacteria have to cope with the accumulation of endogenous reactive oxygen species (ROS). One of the main targets of these ROS is cysteine residues in proteins. This study uses liquid chromatography coupled to high-resolution tandem mass spectrometry to detect significant changes in protein abundance and thiol status for cysteine-containing proteins from Bacillus cereus during aerobic exponential growth. The proteomic profiles of cultures at early-, middle-, and late-exponential growth phases reveals that (i) enrichment in proteins dedicated to fighting ROS as growth progressed, (ii) a decrease in both overall proteome cysteine content and thiol proteome redox status, and (iii) changes to the reduced thiol status of some key proteins, such as the transition state transcriptional regulator AbrB. Taken together, our data indicate that growth under oxic conditions requires increased allocation of protein resources to attenuate the negative effects of ROS. Our data also provide a strong basis to understand the response mechanisms used by B. cereus to deal with endogenous oxidative stress.

Protein Conjugation by Electrophilic Alkynylation Using 5-(Alkynyl)dibenzothiophenium Triflates.

5-(Alkynyl)dibenzothiophenium triflates are introduced as new reagents to prepare different protein conjugates through site-selective cysteine alkynylation. The protocol developed allows a highly efficient label of free cysteine-containing proteins with relevant biological roles, such as ubiquitin, the C2A domain of Synaptotagmin-I, or HER2 targeting nanobodies. An electrophilic bis-alkynylating reagent was also designed. The second alkynylating handle thus introduced in the desired protein enables access to protein-thiol, protein-peptide, and protein-protein conjugates, and even diubiquitin dimers can be prepared through this approach. The low excess of reagent needed, mild reaction conditions used, short reaction times, and stability of the S-C(alkyne) bonds at physiological conditions make this approach an interesting addition to the toolbox of classical, site-selective cysteine-conjugation methods.

Harnessing the potential of bacterial oxidative folding to aid protein production.

Protein folding is central to both biological function and recombinant protein production. In bacterial expression systems, which are easy to use and offer high protein yields, production of the protein of interest in its native fold can be hampered by the limitations of endogenous posttranslational modification systems. Disulfide bond formation, entailing the covalent linkage of proximal cysteine amino acids, is a fundamental posttranslational modification reaction that often underpins protein stability, especially in extracytoplasmic environments. When these bonds are not formed correctly, the yield and activity of the resultant protein are dramatically decreased. Although the mechanism of oxidative protein folding is well understood, unwanted or incorrect disulfide bond formation often presents a stumbling block for the expression of cysteine-containing proteins in bacteria. It is therefore important to consider the biochemistry of prokaryotic disulfide bond formation systems in the context of protein production, in order to take advantage of the full potential of such pathways in biotechnology applications. Here, we provide a critical overview of the use of bacterial oxidative folding in protein production so far, and propose a practical decision-making workflow for exploiting disulfide bond formation for the expression of any given protein of interest.

Electrochemical Investigations of L-Cysteine Interactions with Bismuth Ions

The interaction of L-cysteine with bismuth compounds bismuth(III) salicylate, bismuth(III) citrate, and bismuth(III) nitrate, was studied at pH 1.0 (0.100 M HNO3 and 0.100 M HCl) and pH 7.4 MOPS buffer by cyclic voltammetry at glassy carbon and boron-doped diamond electrodes. pH 1.0, at which bismuth (III) exists as the simple Bi3+ ion, was chosen to approximate the acid strength of stomach contents. pH 7.4, at which bismuth(III) exists as BiO, was used for its similarity to general physiological conditions. The amino acid L-cysteine was chosen because its sulfhydryl group undergoes intense interaction with many metal cations, serving as a model for cysteine-containing proteins in the digestive system. It was determined that Bi(III) and L-cysteine (Cys) form soluble complexes at both pH 1.0 and pH 7.4. UV–vis spectroscopic investigations support interaction of Bi(III) and L-cysteine to form a 1:2 Bi(III): Cys complex in pH 7.4 MOPS buffer. L-cysteine addition to solutions of the pharmaceutical bismuth(III) salicylate was found to alter the voltammetric behavior of the salicylate complex. These results, especially at pH 1.0, are relevant to understanding the interaction of various cysteine-containing proteins in the human digestive system with bismuth pharmaceuticals and may help guide future explorations of bismuth formulations.

Heterogeneous adaptation of cysteine reactivity to a covalent oncometabolite

An important context in which metabolism influences tumorigenesis is the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a disease in which mutation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) causes hyperaccumulation of fumarate. This electrophilic oncometabolite can alter gene activity at the level of transcription, via reversible inhibition of epigenetic dioxygenases, as well as posttranslationally, via covalent modification of cysteine residues. To better understand the potential for metabolites to influence posttranslational modifications important to tumorigenesis and cancer cell growth, here we report a chemoproteomic analysis of a kidney-derived HLRCC cell line. Using a general reactivity probe, we generated a data set of proteomic cysteine residues sensitive to the reduction in fumarate levels caused by genetic reintroduction of active FH into HLRCC cell lines. This revealed a broad up-regulation of cysteine reactivity upon FH rescue, which evidence suggests is caused by an approximately equal proportion of transcriptional and posttranslational modification-mediated regulation. Gene ontology analysis highlighted several new targets and pathways potentially modulated by FH mutation. Comparison of the new data set with prior studies highlights considerable heterogeneity in the adaptive response of cysteine-containing proteins in different models of HLRCC. This is consistent with emerging studies indicating the existence of cell- and tissue-specific cysteine-omes, further emphasizing the need for characterization of diverse models. Our analysis provides a resource for understanding the proteomic adaptation to fumarate accumulation and a foundation for future efforts to exploit this knowledge for cancer therapy.

Characterization of the nonheme iron center of cysteamine dioxygenase and its interaction with substrates

Cysteamine dioxygenase (ADO) has been reported to exhibit two distinct biological functions with a nonheme iron center. It catalyzes oxidation of both cysteamine in sulfur metabolism and N-terminal cysteine-containing proteins or peptides, such as regulator of G protein signaling 5 (RGS5). It thereby preserves oxygen homeostasis in a variety of physiological processes. However, little is known about its catalytic center and how it interacts with these two types of primary substrates in addition to O2 Here, using electron paramagnetic resonance (EPR), Mössbauer, and UV-visible spectroscopies, we explored the binding mode of cysteamine and RGS5 to human and mouse ADO proteins in their physiologically relevant ferrous form. This characterization revealed that in the presence of nitric oxide as a spin probe and oxygen surrogate, both the small molecule and the peptide substrates coordinate the iron center with their free thiols in a monodentate binding mode, in sharp contrast to binding behaviors observed in other thiol dioxygenases. We observed a substrate-bound B-type dinitrosyl iron center complex in ADO, suggesting the possibility of dioxygen binding to the iron ion in a side-on mode. Moreover, we observed substrate-mediated reduction of the iron center from ferric to the ferrous oxidation state. Subsequent MS analysis indicated corresponding disulfide formation of the substrates, suggesting that the presence of the substrate could reactivate ADO to defend against oxidative stress. The findings of this work contribute to the understanding of the substrate interaction in ADO and fill a gap in our knowledge of the substrate specificity of thiol dioxygenases.