H2S, Polysulfides, and Enzymes: Physiological and Pathological Aspects.

We have been studying the general aspects of the functions of H2S and polysulfides, and the enzymes involved in their biosynthesis, for more than 20 years. Our aim has been to elucidate novel physiological and pathological functions of H2S and polysulfides, and unravel the regulation of the enzymes involved in their biosynthesis, including cystathionine β-synthase (EC 4.2.1.22), cystathionine γ-lyase (EC 4.4.1.1), thiosulfate sulfurtransferase (rhodanese, EC 2.8.1.1), and 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2). Physiological and pathological functions, alternative biosynthetic processes, and additional functions of H2S and polysulfides have been reported. Further, the structure and reaction mechanisms of related enzymes have also been reported. We expect this issue to advance scientific knowledge regarding the detailed functions of H2S and polysulfides as well as the general properties and regulation of the enzymes involved in their metabolism. We would like to cover four topics: the physiological and pathological functions of H2S and polysulfides, the mechanisms of the biosynthesis of H2S and polysulfides, the properties of the biosynthetic enzymes, and the regulation of enzymatic activity. The knockout mouse technique is a useful tool to determine new physiological functions, especially those of H2S and polysulfides. In the future, we shall take a closer look at symptoms in the human congenital deficiency of each enzyme. Further studies on the regulation of enzymatic activity by in vivo substances may be the key to finding new functions of H2S and polysulfides.

On the other hand, Nagahara et al. [11] recently demonstrated in vitro that MST transfers a sulfur atom from 3-mercptopyruvate (MP) to the catalytic site cysteine to form stable persulfide (polysulfide) as a reaction intermediate. It is interesting that as an alternative production process, thiol-containing compounds attack the persulfide (polysulfide) formed at the catalytic site and a new persulfide (polysulfide) molecule is formed at the thiol-containing compound. Then, dithiol is reduced by thioredoxin (Trx) or dihydrolipoic acid to release H 2 S or polysufides. This process may be autoreduction. Yadav et al. [10] performed enzyme kinetics analysis for human MST in the production process of hydrogen disulfide.
To understand the physiological function of sulfane sulfur, its levels in biological samples (tissues, cell cultures) were determined using the reaction with cyanide, and subsequently investigating the thiocyanate yield of the complex with Fe 3+ , which is detectable by spectrophotometry [35]. Although the method is not very sensitive, it showed that sulfane sulfur levels were quite similar in various animal tissues [36,37] and in murine macrophages despite stimulation with lipopolysaccharide and interferon-γ [38]. Thus, an argument may be put forward about homeostasis of sulfane sulfur levels in biological systems. Moreover, a negative feedback regulation between CBS and CTH was suggested by Nandi and Mishra [39] and confirmed by Bronowicka et al. [38]. The adaptive cellular response to stimulation with both IFNγ and LPS caused a decreased level of H 2 S-associated low CBS expression and increased CTH expression. The adaptive cellular response to electrophiles (electron-deficient species) represented by heavy metal ions [40] involves sulfane sulfur atoms of numerous SSBPs. Protection against electrophilic stresses involves persulfides rather than thiol groups because of their higher nucleophilicity [41]. Electrophiles are captured by reactive persulfide/polysulfide species, resulting in formation of their sulfur adducts [42]. In bovine aortic endothelial cells, CSE knockdown potentiated Cd-induced cytotoxicity but CSE overexpression provided protection [43]. In vivo experiments showed that CSE-knockout mice were sensitive to Cd-induced hepatotoxicity [44]. Adaptive changes in the activity and expression of CGL, MST, and TST in various frog tissues in response to exposure to lead, mercury, and cadmium confirmed the protective function of these enzymatic proteins against electrophilic stress [45,46].

Knockout of H 2 S and Polysulfides-Producing Enzymes
Knockout (KO) technique is a good tool to clarify the physiological functions of proteins. Congenial deficiency of CBS causes hyperhomocysteinemia or homocystinuria in humans. Watanabe et al. produced CBS-KO mice [52], and the mice were afflicted with chronic renal dysfunction. The mice showed growth retardation and died within 5 weeks.
Congenial deficiency of CGL causes cystathioninuria in humans. CGL-KO mice were produced by Yang et al. [53], and the mice displayed low levels of H 2 S associated with hypertension.
Congenial deficiency of MST causes mercaptolactate-cysteine disulfidria in humans. MST-KO mice were produced by Nagahara et al. [54]; however, mercaptolactate-cysteine disulfiduria has not been examined. Peleli et al. [55] recently reported that MST-KO mice were protected against ischemic reperfusion of the heart. Nasi et al. [56] reported that mice showed accelerated joint calcification and osteoarthritis due to an increase in chondrocyte mineralization. Interestingly, these two findings indicate that MST demonstrates both good and bad effects on living organisms.
TST-KO mice have not been obtained; however, a double KO mouse for both TST and MST has been produced (unpublished data).

Conclusions
Four cysteine-containing enzymes (CBS, CGL, MST, and TST) produce H 2 S and polysulfides via the reduction of persulfurated or polysulfurated substrates. MST is also produced via the reduction of stable persulfide (polysulfide) formed at a catalytic-site cysteine as a reaction intermediate. Sulfur oxide can also be produced from a catalytic site cysteine of MST. These products play an important role in living organisms. Furthermore, studies using KO mice of each enzyme have clarified the physiological function of these enzymes, which cannot be assessed in wild-type animals. Regulation of enzymatic activity by in vivo substances may be related to the functions of H 2 S and polysulfides.