An official website of the United States government
Bacillithiol (BSH) replaces glutathione (GSH) as the most prominent low-molecular-weight thiol in many low G + C gram-positive bacteria. BSH plays roles in metal binding, protein/ enzyme regulation, detoxification, redox buffering, and bacterial virulence. Given the small amounts of BSH isolated from natural sources and relatively lengthy chemical syntheses, the reactions of BSH with pertinent reactive oxygen, nitrogen, and sulfur species remain largely unexplored. We prepared BSH and exposed it to nitroxyl (HNO), a reactive nitrogen species that influences bacterial sulfur metabolism. The profile of this reaction was distinct from HNO oxidation of GSH, which yielded mixtures of disulfide and sulfinamide. The reaction of BSH and HNO (generated from Angeli’s salt) gives only sulfinamide products, including a newly proposed cyclic sulfinamide. Treatment of a glucosamine−cysteine conjugate, which lacks the malic acid group, with HNO forms disulfide, implicating the malic acid group in sulfinamide formation. This finding supports a mechanism involving the formation of an N-hydroxysulfenamide intermediate that dehydrates to a sulfenium ion that can be trapped by water or internally trapped by an amide nitrogen to give the cyclic sulfinamide. The biological relevance of BSH reactivity toward HNO is provided through in vivo experiments demonstrating that Bacillus subtilis exposed to HNO shows a growth phenotype, and a strain unable to produce BSH shows hypersensitivity toward HNO in minimal medium cultures. Thiol analysis of HNO-exposed cultures shows an overall decrease in reduced BSH levels, which is not accompanied by increased levels of BSSB, supporting a model involving the formation of an oxidized sulfinamide derivative, identified in vivo by high-pressure liquid chromatography/mass spectrometry. Collectively, these findings reveal the unique chemistry and biology of HNO with BSH in bacteria that produce this biothiol.
more »
« less
This content will become publicly available on December 1, 2026
Differential reactivity of bacillithiol, mycothiol and glutathione with nitroxyl (HNO): Structural determinants and potential biological implications
Low-molecular-weight (LMW) thiols play critical roles in maintaining redox buffer systems required for normal biological function. Glutathione (GSH) represents the most common LMW thiol found in Nature, but Gram-positive bacteria utilize bacillithiol (BSH) or mycothiol (MSH). Nitroxyl (HNO) can influence bacterial transcription through persulfide formation, a biological phenomenon that prompts the examination of the reactions of HNO with these LMW thiols. The development and application of colorimetric and enzymatic (Bacillus subtilis thioredoxin assay) methods combined with mass spectrometry of reaction products show the unique reactivity of BSH to favor sulfinamide adduct formation upon equimolar reaction with HNO. The reaction profile with GSH results in nearly equal distribution between sulfinamide:disulfide, whereas reaction with MSH only yields disulfide. These varied results led to the preparation of a group of BSH and MSH analogs, and their reactions with HNO reveal the requirement for a free amine group for sulfinamide formation. The thiol and amine group pKa's appear critical for sulfinamide generation, with the thiolate acting as a nucleophile to attack HNO and the ammonium donating a proton to facilitate water loss from the N-hydroxysulfenamide intermediate. Furthermore, the B. subtilis thioredoxin system efficiently reduces BSSB with a calculated KM_BSSB = 34 ± 3 μM and Vmax = 152 ± 3.4 nmol/min/nmol TrxR (kcat = 2.5 s−1), but does not reduce bacillithiol sulfinamide. Similarly, this thioredoxin reduces MSSM with a calculated KM_MSSM of 9 ± 2.1 μM and Vmax of 103 ± 7.1 nmol/min/nmol TrxR (kcat = 1.7 s−1). Bacillithiol possesses a unique structure that allows a rapid reaction with HNO to form a stable product that may provide a basis for antibiotic development or clues for further biological roles of nitroxyl.
more »
« less
- Award ID(s):
- 1716535
- PAR ID:
- 10659171
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Redox Biology
- Volume:
- 88
- Issue:
- C
- ISSN:
- 2213-2317
- Page Range / eLocation ID:
- 103915
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Naphthoquinone (1,4-NQ) and its derivatives (NQs, juglone, plumbagin, 2-methoxy-1,4-NQ, and menadione) have a variety of therapeutic applications, many of which are attributed to redox cycling and the production of reactive oxygen species (ROS). We previously demonstrated that NQs also oxidize hydrogen sulfide (H2S) to reactive sulfur species (RSS), potentially conveying identical benefits. Here we use RSS-specific fluorophores, mass spectroscopy, EPR and UV-Vis spectrometry, and oxygen-sensitive optodes to examine the effects of thiols and thiol-NQ adducts on H2S-NQ reactions. In the presence of glutathione (GSH) and cysteine (Cys), 1,4-NQ oxidizes H2S to both inorganic and organic hydroper-/hydropolysulfides (R2Sn, R=H, Cys, GSH; n = 2–4) and organic sulfoxides (GSnOH, n = 1, 2). These reactions reduce NQs and consume oxygen via a semiquinone intermediate. NQs are also reduced as they form adducts with GSH, Cys, protein thiols, and amines. Thiol, but not amine, adducts may increase or decrease H2S oxidation in reactions that are both NQ- and thiol-specific. Amine adducts also inhibit the formation of thiol adducts. These results suggest that NQs may react with endogenous thiols, including GSH, Cys, and protein Cys, and that these adducts may affect both thiol reactions as well as RSS production from H2S.more » « less
-
null (Ed.)Developing an engineerable chemical reaction that is triggerable for simultaneous chemical bond formation and cleavage by external cues offers tunability and orthogonality which is highly desired in many biological and materials applications. Here, we present a chemical switch that concurrently captures these features in response to chemically and biologically abundant and important cues, viz. , thiols and amines. This thiol/amine-triggerable chemical switch is based on a Triggerable Michael Acceptor (TMAc) which bears good leaving groups at its β-position. The acceptor undergoes a “trigger-to-release” process where thiol/amine addition triggers cascaded release of leaving groups and generates a less activated acceptor. The newly generated TMAc can be further reversed to liberate the original thiol/amine by a second nucleophile trigger through a “trigger-to-reverse” process. Within the small molecular volume of the switch, we have shown five locations that can be engineered to achieve tunable “trigger-to-release” kinetics and tailored reversibility. The potential of the engineerable bonding/debonding capability of the chemical switch is demonstrated by applications in cysteine-selective and reversible protein modification, universal self-immolative linkers, and orthogonally addressable hydrogels.more » « less
-
Thioredoxin reductases (TrxR) activate thioredoxins (Trx) that regulate the activity of diverse target proteins essential to prokaryotic and eukaryotic life. However, very little is understood of TrxR/Trx systems and redox control in methanogenic microbes from the domain Archaea (methanogens), for which genomes are abundant with annotations for ferredoxin:thioredoxin reductases [Fdx/thioredoxin reductase (FTR)] from group 4 of the widespread FTR-like family. Only two from the FTR-like family are characterized: the plant-type FTR from group 1 and FDR from group 6. Herein, the group 4 archetype (AFTR) from Methanosarcina acetivorans was characterized to advance understanding of the family and TrxR/Trx systems in methanogens. The modeled structure of AFTR, together with EPR and Mössbauer spectroscopies, supports a catalytic mechanism similar to plant-type FTR and FDR, albeit with important exceptions. EPR spectroscopy of reduced AFTR identified a transient [4Fe−4S]1+ cluster exhibiting a mixture of S = 7/2 and typical S = 1/2 signals, although rare for proteins containing [4Fe−4S] clusters, it is most likely the on-pathway intermediate in the disulfide reduction. Furthermore, an active site histidine equivalent to residues essential for the activity of plant-type FTR and FDR was found dispensable for AFTR. Finally, a unique thioredoxin system was reconstituted from AFTR, ferredoxin, and Trx2 from M. acetivorans, for which specialized target proteins were identified that are essential for growth and other diverse metabolisms.more » « less
-
null (Ed.)The acid-catalyzed thiol–ene reaction (ACT) is a unique thiol–X conjugation strategy that produces S,X-acetal conjugates. Unlike the well-known radical-mediated thiol–ene and anion-mediated thiol-Michael reactions that produce static thioether bonds, acetals provide unique function for various fields such as drug delivery and protecting group chemistries; however, this reaction is relatively underutilized for creating new and unique materials owing to the unexplored reactivity over a broad set of substrates and potential side reactions. Solution-phase studies using a range of thiol and alkene substrates were conducted to evaluate the ACT reaction as a conjugation strategy. Substrates that efficiently undergo cationic polymerizations, such as those containing vinyl functional groups, were found to be highly reactive to thiols in the presence of catalytic amounts of acid. Additionally, sequential initiation of three separate thiol–X reactions (thiol-Michael, ACT, and thiol–ene) was achieved in a one-pot scheme simply by the addition of the appropriate catalyst demonstrating substrate selectivity. Furthermore, photoinitiation of the ACT reaction was achieved for the first time under 470 nm blue light using a novel photochromic photoacid. Finally, using multifunctional monomers, solid-state polymer networks were formed using the ACT reaction producing acetal crosslinks. The presence of S,X-acetal bonds results in an increased glass transition temperature of 20 °C as compared with the same polymeric film polymerized through the radical thiol–ene mechanism. This investigation demonstrates the broad impact of the ACT reaction and expands upon the diverse thiol–X library of conjugation strategies towards the development of novel materials systems.more » « less
