Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?

BACKGROUND AND PURPOSE: Posttranslational modifications of cysteine residues represent a major aspect of redox biology, and their reliable detection is key in providing mechanistic insights. The metastable character of these modifications and cell lysis-induced artifactual oxidation render current state-of-the-art protocols to rely on alkylation-based stabilization of labile cysteine derivatives before cell/tissue rupture. An untested assumption in these procedures is that for all cysteine derivatives, alkylation rates are faster than their dynamic interchange. However, when the interconversion of cysteine derivatives is not rate limiting, electrophilic labelling is under Curtin-Hammett control; hence, the final alkylated mixture may not represent the speciation that prevailed before alkylation. EXPERIMENTAL APPROACH: Buffered aqueous solutions of inorganic, organic, cysteine, GSH and GAPDH polysulfide species were used. Additional experiments in human plasma and serum revealed that monobromobimane can extract sulfide from the endogenous sulfur pool by shifting speciation equilibria, suggesting caution should be exercised when interpreting experimental results using this tool. KEY RESULTS: In the majority of cases, the speciation of alkylated polysulfide/thiol derivatives depended on the experimental conditions. Alkylation perturbed sulfur speciation in both a concentration- and time-dependent manner and strong alkylating agents cleaved polysulfur chains. Moreover, the labelling of sulfenic acids with dimedone also affected cysteine speciation, suggesting that part of the endogenous pool of products previously believed to represent sulfenic acid species may represent polysulfides. CONCLUSIONS AND IMPLICATIONS: We highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiological speciation of sulfur species. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Kinetic Insights into Hydrogen Sulfide Delivery from Caged-Carbonyl Sulfide Isomeric Donor Platforms.

Hydrogen sulfide (H2S) is a biologically important small gaseous molecule that exhibits promising protective effects against a variety of physiological and pathological processes. To investigate the expanding roles of H2S in biology, researchers often use H2S donors to mimic enzymatic H2S synthesis or to provide increased H2S levels under specific circumstances. Aligned with the need for new broad and easily modifiable platforms for H2S donation, we report here the preparation and H2S release kinetics from a series of isomeric caged-carbonyl sulfide (COS) compounds, including thiocarbamates, thiocarbonates, and dithiocarbonates, all of which release COS that is quickly converted to H2S by the ubiquitous enzyme carbonic anhydrase. Each donor is designed to release COS/H2S after the activation of a trigger by activation by hydrogen peroxide (H2O2). In addition to providing a broad palette of new, H2O2-responsive donor motifs, we also demonstrate the H2O2 dose-dependent COS/H2S release from each donor core, establish that release profiles can be modified by structural modifications, and compare COS/H2S release rates and efficiencies from isomeric core structures. Supporting our experimental investigations, we also provide computational insights into the potential energy surfaces for COS/H2S release from each platform. In addition, we also report initial investigations into dithiocarbamate cores, which release H2S directly upon H2O2-mediated activation. As a whole, the insights on COS/H2S release gained from these investigations provide a foundation for the expansion of the emerging area of responsive COS/H2S donor systems.

The Intersection of NO and H2S: Persulfides Generate NO from Nitrite through Polysulfide Formation.

Hydrogen sulfide (H2S) and nitric oxide (NO) are important biosignaling molecules, and their biochemistries are increasingly recognized to be intertwined. Persulfides are an oxidized product of biological H2S and have emerged as important species involved in the biological action of reactive sulfur species. Using isolated persulfides, we employed a combination of experimental and computational methods to investigate the contribution of persulfides to H2S/NO crosstalk. Our studies demonstrate that isolated persulfides react with nitrite to produce NO via polysulfide and perthionitrite intermediates. These results highlight the importance of persulfides, polysulfides, and perthionitrite as intertwined reactive nitrogen and sulfur species.

Mechanistic Insights into the H2S-Mediated Reduction of Aryl Azides Commonly Used in H2S Detection.

Hydrogen sulfide (H2S) is an important biological mediator and has been at the center of a rapidly expanding field focused on understanding the biogenesis and action of H2S as well as other sulfur-related species. Concomitant with this expansion has been the development of new chemical tools for H2S research. The use of H2S-selective fluorescent probes that function by H2S-mediated reduction of fluorogenic aryl azides has emerged as one of the most common methods for H2S detection. Despite this prevalence, the mechanism of this important reaction remains under-scrutinized. Here we present a combined experimental and computational investigation of this mechanism. We establish that HS(-), rather than diprotic H2S, is the active species required for aryl azide reduction. The hydrosulfide anion functions as a one-electron reductant, resulting in the formation of polysulfide anions, such as HS2(-), which were confirmed and trapped as organic polysulfides by benzyl chloride. The overall reaction is first-order in both azide and HS(-) under the investigated experimental conditions with ΔS(⧧) = -14(2) eu and ΔH(⧧) = 13.8(5) kcal/mol in buffered aqueous solution. By using NBu4SH as the sulfide source, we were able to observe a reaction intermediate (λ(max) = 473 nm), which we attribute to formation of an anionic azidothiol intermediate. Our mechanistic investigations support that this intermediate is attacked by HS(-) in the rate-limiting step of the reduction reaction. Complementing our experimental mechanistic investigations, we also performed DFT calculations at the B3LYP/6-31G(d,p), B3LYP/6-311++G(d,p), M06/TZVP, and M06/def2-TZVPD levels of theory applying the IEF-PCM water and MeCN solvation models, all of which support the experimentally determined reaction mechanism and provide cohesive mechanistic insights into H2S-mediated aryl azide reduction.