The Rhodanese PspE Converts Thiosulfate to Cellular Sulfane Sulfur in Escherichia coli.

Hydrogen sulfide (H2S) and its oxidation product zero-valent sulfur (S0) play important roles in animals, plants, and bacteria. Inside cells, S0 exists in various forms, including polysulfide and persulfide, which are collectively referred to as sulfane sulfur. Due to the known health benefits, the donors of H2S and sulfane sulfur have been developed and tested. Among them, thiosulfate is a known H2S and sulfane sulfur donor. We have previously reported that thiosulfate is an effective sulfane sulfur donor in Escherichia coli; however, it is unclear how it converts thiosulfate to cellular sulfane sulfur. In this study, we showed that one of the various rhodaneses, PspE, in E. coli was responsible for the conversion. After the thiosulfate addition, the ΔpspE mutant did not increase cellular sulfane sulfur, but the wild type and the complemented strain ΔpspE::pspE increased cellular sulfane sulfur from about 92 µM to 220 µM and 355 µM, respectively. LC-MS analysis revealed a significant increase in glutathione persulfide (GSSH) in the wild type and the ΔpspE::pspE strain. The kinetic analysis supported that PspE was the most effective rhodanese in E. coli in converting thiosulfate to glutathione persulfide. The increased cellular sulfane sulfur alleviated the toxicity of hydrogen peroxide during E. coli growth. Although cellular thiols might reduce the increased cellular sulfane sulfur to H2S, increased H2S was not detected in the wild type. The finding that rhodanese is required to convert thiosulfate to cellular sulfane sulfur in E. coli may guide the use of thiosulfate as the donor of H2S and sulfane sulfur in human and animal tests.

Optimization of a Method for Detecting Intracellular Sulfane Sulfur Levels and Evaluation of Reagents That Affect the Levels in Escherichia coli.

Sulfane sulfur is a class of compounds containing zero-valent sulfur. Most sulfane sulfur compounds are reactive and play important signaling roles. Key enzymes involved in the production and metabolism of sulfane sulfur have been characterized; however, little is known about how to change intracellular sulfane sulfur (iSS) levels. To accurately measure iSS, we optimized a previously reported method, in which reactive iSS reacts with sulfite to produce thiosulfate, a stable sulfane sulfur compound, before detection. With the improved method, several factors were tested to influence iSS in Escherichia coli. Temperature, pH, and osmotic pressure showed little effect. At commonly used concentrations, most tested oxidants, including hydrogen peroxide, tert-butyl hydroperoxide, hypochlorous acid, and diamide, did not affect iSS, but carbonyl cyanide m-chlorophenyl hydrazone increased iSS. For reductants, 10 mM dithiothreitol significantly decreased iSS, but tris(2-carboxyethyl)phosphine did not. Among different sulfur-bearing compounds, NaHS, cysteine, S2O32- and diallyl disulfide increased iSS, of which only S2O32- did not inhibit E. coli growth at 10 mM or less. Thus, with the improved method, we have identified reagents that may be used to change iSS in E. coli and other organisms, providing tools to further study the physiological functions of iSS.

Rhodaneses minimize the accumulation of cellular sulfane sulfur to avoid disulfide stress during sulfide oxidation in bacteria.

Heterotrophic bacteria and human mitochondria often use sulfide: quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) to oxidize sulfide to sulfite and thiosulfate. Bioinformatic analysis showed that the genes encoding RHOD domains were widely presented in annotated sqr-pdo operons and grouped into three types: fused with an SQR domain, fused with a PDO domain, and dissociated proteins. Biochemical evidence suggests that RHODs facilitate the formation of thiosulfate and promote the reaction between inorganic polysulfide and glutathione to produce glutathione polysulfide. However, the physiological roles of RHODs during sulfide oxidation by SQR and PDO could only be tested in an RHOD-free host. To test this, 8 genes encoding RHOD domains in Escherichia coli MG1655 were deleted to produce E. coli RHOD-8K. The sqrCp and pdoCp genes from Cupriavidus pinatubonensis JMP134 were cloned into E. coli RHOD-8K. SQRCp contains a fused RHOD domain at the N-terminus. When the fused RHOD domain of SQRCp was inactivated, the cells oxidized sulfide into increased thiosulfate with the accumulation of cellular sulfane sulfur in comparison with cells containing the intact sqrCp and pdoCp. The complementation of dissociated DUF442 minimized the accumulation of cellular sulfane sulfur and reduced the production of thiosulfate. Further analysis showed that the fused DUF442 domain modulated the activity of SQRCp and prevented it from directly passing the produced sulfane sulfur to GSH. Whereas, the dissociated DUF442 enhanced the PDOCp activity by several folds. Both DUF442 forms minimized the accumulation of cellular sulfane sulfur, which spontaneously reacted with GSH to produce GSSG, causing disulfide stress during sulfide oxidation. Thus, RHODs may play multiple roles during sulfide oxidation.

The Pathway of Sulfide Oxidation to Octasulfur Globules in the Cytoplasm of Aerobic Bacteria.

Sulfur-oxidizing bacteria can oxidize hydrogen sulfide (H2S) to produce sulfur globules. Although the process is common, the pathway is unclear. In recombinant Escherichia coli and wild-type Corynebacterium vitaeruminis DSM 20294 with sulfide:quinone oxidoreductase (SQR) but no enzymes to oxidize zero valence sulfur, SQR oxidized H2S into short-chain inorganic polysulfide (H2Sn, n ≥ 2) and organic polysulfide (RSnH, n ≥ 2), which reacted with each other to form long-chain GSnH (n ≥ 2) and H2Sn before producing octasulfur (S8), the main component of elemental sulfur. GSnH also reacted with glutathione (GSH) to form GSnG (n ≥ 2) and H2S; H2S was again oxidized by SQR. After GSH was depleted, SQR simply oxidized H2S to H2Sn, which spontaneously generated S8. S8 aggregated into sulfur globules in the cytoplasm. The results highlight the process of sulfide oxidation to S8 globules in the bacterial cytoplasm and demonstrate the potential of using heterotrophic bacteria with SQR to convert toxic H2S into relatively benign S8 globules. IMPORTANCE Our results provide evidence of H2S oxidation producing octasulfur globules via sulfide:quinone oxidoreductase (SQR) catalysis and spontaneous reactions in the bacterial cytoplasm. Since the process is an important event in geochemical cycling, a better understanding facilitates further studies and provides theoretical support for using heterotrophic bacteria with SQR to oxidize toxic H2S into sulfur globules for recovery.

Sensitive Method for Reliable Quantification of Sulfane Sulfur in Biological Samples.

Sulfane sulfur has been recognized as a common cellular component, participating in regulating enzyme activities and signaling pathways. However, the quantification of total sulfane sulfur in biological samples is still a challenge. Here, we developed a method to address the need. All tested sulfane sulfur reacted with sulfite and quantitatively converted to thiosulfate when heated at 95 °C in a solution of pH 9.5 for 10 min. The assay condition was also sufficient to convert total sulfane sulfur in biological samples to thiosulfate for further derivatization and quantification. We applied the method to detect sulfane sulfur contents at different growth phases of bacteria, yeast, mammalian cells, and zebrafish. Total sulfane sulfur contents in all of them increased in the early stage, kept at a steady state for a period, and declined sharply in the late stage of the growth. Sulfane sulfur contents varied in different species. For Escherichia coli, growth media also affected the sulfane sulfur contents. Total sulfane sulfur contents from different organs of mouse and shrimp were also detected, varying from 1 to 10 nmol/(mg of protein). Thus, the new method is suitable for the quantification of total sulfane sulfur in biological samples.

Using resonance synchronous spectroscopy to characterize the reactivity and electrophilicity of biologically relevant sulfane sulfur.

Sulfane sulfur is common inside cells, playing both regulatory and antioxidant roles. However, there are unresolved issues about its chemistry and biochemistry. We report the discovery that reactive sulfane sulfur such as polysulfides and persulfides could be detected by using resonance synchronous spectroscopy (RS2). With RS2, we showed that inorganic polysulfides at low concentrations were unstable with a half-life about 1 min under physiological conditions due to reacting with glutathione. The protonated form of glutathione persulfide (GSSH) was electrophilic and had RS2 signal. GSS- was nucleophilic, prone to oxidation, but had no RS2 signal. Using this phenomenon, pKa of GSSH was determined as 6.9. GSSH/GSS- was 50-fold more reactive than H2S/HS- towards H2O2 at pH 7.4, supporting reactive sulfane sulfur species like GSSH/GSS- may act as antioxidants inside cells. Further, protein persulfides were shown to be in two forms: at pH 7.4 the deprotonated form (R-SS-) without RS2 signal was not reactive toward sulfite, and the protonated form (R-SSH) in the active site of a rhodanese had RS2 signal and readily reacted with sulfite to produce thiosulfate. These data suggest that RS2 of sulfane sulfur is likely associated with its electrophilicity. Sulfane sulfur showed species-specific RS2 spectra and intensities at physiological pH, which may reveal the relative abundance of a reactive sulfane sulfur species inside cells.