Millions of years ago, life on Earth emerged in an H2S-rich environment [1], flourishing and evolving with H2S-mediated signaling mechanisms. Sulfur-containing molecules, including amino acids, may have been formed from H2S released by volcanic emissions and geothermal activity under atmospheric conditions on early Earth [[2], [3], [4]]. In recent years, H2S, has re-emerged in life sciences as the third gaseous signaling molecule, and its recognized signaling mechanism is the persulfidation of cysteine residues [[5], [6], [7], [8], [9], [10]]. It was reported [6,7,11,12] that H2S may convert cysteinyl thiolates (Cys-S-) to persulfides (Cys-S-S-) through sulfane (Sn0, a sulfur-containing intermediate). Thus, dihydrogen sulfane (H2Sn) is now the names recommended for H2S by International Union of Pure and Applied Chemistry (IUPAC) [6].

Glutathione (GSH) is a functional short peptide composed of three amino acids: glutamic acid (Glu), cysteine (Cys), and glycine (Gly) [13]. GSH is involved in many biological functions, such as maintaining intracellular redox states [14], stabilizing the cholinergic system [15,16], intracellular signal transduction [17,18], and gene regulation [[19], [20], [21]]. In a previous study, we reported that GSH could react with the free sulfhydryl groups in enzymes to generate protein-SG or protein-SSG derivatives by S-desulfurization (a modification that removes the sulfur to release H2S, as opposed to persulfidation in the redox state), thereby regulating enzyme protein activity [12]. We elucidated the GSH and H2Sn mechanisms in a free sulfhydryl system without involving a disulfide structure [12]. Little is known about whether GSH and H2Sn can modify the oxidized sulfhydryl structure of the disulfide bonds of proteins.

Disulfide bonds are important post-translational modifications in proteins within the endoplasmic reticulum, playing a crucial role in maintaining structure and stability [22]. They act as redox catalytic groups or allosteric switches in enzymes, governing protein function [23]. To explore the biological significance of disulfide bonds in enzymes, we chose AChE as a model protease. Acetylcholinesterase (AChE) is a serine hydrolase responsible for degrading acetylcholine neurotransmitters into acetate and choline [24,25]. AChE plays a vital role in maintaining the neurotransmitter acetylcholine (ACh) levels in the cholinergic system. It was found that the molecular mechanisms of the pathogenesis of some diseases are related to overactivation of AChE, such as epilepsy, Alzheimer's disease [26], Parkinson's disease [27], and depression [28]. The advantage of choosing AChE (Electrophorus electricus) is that it is a mature commercial enzyme with a complete and identifiable amino acid sequence and multiple identifiable catalytic domains. More importantly, there are six cysteine residues in AChE that form three sets of disulfide bonds without free sulfhydryl groups, which is conducive to investigating whether the modification caused by H2Sn or GSH can occur in the case of disulfide bonds. The three disulfide bonds in AChE are S-S① (Cys69-Cys96), S-S② (Cys257-Cys272), and S-S③ (Cys409-Cys529). In our previous report, it has been confirmed that diallyl trisulfide (DATS) derived from Allium irreversibly inhibited AChE activity by opening disulfide-bond switching of S-S① (Cys69-Cys96) and S-S② (Cys257-Cys272) in AChE, as well as by covalently modifying Cys-272 in S-S②to generate modification containing allyl sulfur to strengthen switch [23]. To further clarify the biological significance of the disulfide bond switch on the catalytic functions involved in the structure of AChE, especially on the function of “catalytic canyon”, a reaction cavity for a catalytic substrate, we present our insights based on a re-exploration of “self-breathing motion” in AChE during substrate catalytic process.

The aim of this study is to explore the mechanisms of two representative sulfides (H2Sn and GSH, with different sulfhydryl modification mechanisms) on disulfide bonds, as well as the influence of disulfide-bond switching states on “catalytic canyon”, to enrich and improve the catalytic theory of AChE. Here, we provide direct evidences via matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) that H2Sn and GSH can open certain disulfide bonds (nucleophilic attack) and persulfidate (induced by H2Sn) or glutathionylate (induced by GSH) the specific thiol to generate protein-SSH or protein-SSG, respectively. The chemical formation mechanisms of protein-SSH and protein-SSG were also elaborated in detail. Using molecular flexible docking technology combined with stoichiometry, the differences between H2Sn and GSH in opening disulfide bonds in different chemical environments of the protein were shown. In the catalysis of AChE simulated by molecular dynamics (MD), the process of AChE hydrolysis of ACh was similar to a kind of “self-breathing motion” [[29], [30], [31]]. A complete 10 ns “self-breathing motion” was successfully acquired and displayed in this paper. Combined with the results of circular dichroism (CD), mass spectrometry, molecular docking, stoichiometry, and MD simulations, we proposed and summarized the “theory of self-breathing regulation” derived by H2Sn and GSH on controlling AChE activity. We also found that NaHS, K2Sn, and GSH prevented of overactivation of AChE, and exhibited anti-memory-impairment, anti-anxiety and anti-depressant effects in a lipopolysaccharide (LPS)-induced mouse model. These findings and theoretical elaborations complement the mechanisms by which H2Sn and GSH act as signaling molecules in vivo in response to protein disulfide bonds conditions. It also provides a theoretical basis for the treatment of AChE-related diseases and the development of enzyme inhibitors targeting disulfide bonds.