Saccharomyces cerevisiae is known to grow with thiosulfate as a sulfur source, and it produces more ethanol when using thiosulfate than using sulfate. Here we report how it assimilates thiosulfate. S. cerevisiae absorbed thiosulfate into the cell through two sulfate permeases, Sul1 and Sul2. Two rhodaneses, Rdl1 and Rdl2, converted thiosulfate to a persulfide and sulfite. The persulfide was reduced by cellular thiols to H₂S, and sulfite was reduced by sulfite reductase to H₂S. Cysteine synthase incorporated H₂S into O-acetyl-L-homoserine to produce L-homocysteine, which is the precursor for cysteine and methionine in S. cerevisiae. Several other rhodaneses replaced Rdl1 and Rdl2 for thiosulfate utilization in the yeast. Thus, any organisms with the sulfate assimilation system could potentially use thiosulfate as a sulfur source since rhodaneses are common in most organisms. Importance The complete pathway of thiosulfate assimilation in the baker9s yeast is determined. The finding reveals the extensive overlap between sulfate and thiosulfate assimilation. Rhodanese is the only additional enzyme for thiosulfate utilization. The common presence of rhodanese in most organisms, including Bacteria, Archaea, and Eukarya, suggests that most organisms with the sulfate assimilation system may also use thiosulfate. Since it takes less energy to reduce thiosulfate than sulfate for assimilation, thiosulfate has the potential to become a choice of sulfur in optimized media for industrial fermentation.

Elemental sulfur and sulfite have been used to inhibit the growth of yeasts, but thiosulfate has not been reported to be toxic to yeasts. We observed that thiosulfate was more inhibitory than sulfite to Saccharomyces cerevisiae growing in a common yeast medium. At pH < 4, thiosulfate was a source of elemental sulfur and sulfurous acid, and both were highly toxic to the yeast. At pH 6, thiosulfate directly inhibited the electron transport chain in yeast mitochondria, leading to reductions in oxygen consumption, mitochondrial membrane potential and cellular ATP. Although thiosulfate was converted to sulfite and H₂S by the mitochondrial rhodanese Rdl1, its toxicity was not due to H₂S as the rdl1-deletion mutant that produced significantly less H₂S was more sensitive to thiosulfate than the wild type. Evidence suggests that thiosulfate inhibits cytochrome c oxidase of the electron transport chain in yeast mitochondria. Thus, thiosulfate is a potential agent against yeasts.

During aerobic respiration, mitochondria generate superoxide anion (O ₂ ^•− ), hydrogen peroxide (H ₂ O ₂ ), and hydroxyl radical (HO ^• ), and these reactive oxygen species (ROS) are detrimental to mitochondria. Mitochondrial damage is linked to a broad spectrum of pathologies such as Alzheimer’s disease, hemochromatosis, and diabetes. Mitochondria contain several enzymes for rapidly removing superoxide anion and hydrogen peroxide, but how they antagonize HO ^• is elusive, representing a loophole in the anti-ROS system. Herein, we discovered that Rhodanese 2 (Rdl2) is critical for maintaining the functionality and integrity of mitochondria under sub-lethal ROS stress in Saccharomyces cerevisiae . Rdl2 converts stable sulfur species (thiosulfate and dialkyl polysulfide) to reactive sulfane sulfur including persulfide that protects mitochondrial DNA via scavenging HO ^• . Surprisingly, hydrogen sulfide (H ₂ S) promotes HO ^• production through stimulating the Fenton reaction, leading to increased DNA damage. Our study may reveal an ex-ante mean for antagonizing HO ^• , patching the loophole of the anti-ROS system in mitochondria.