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Hydrogen sulfide (H 2 S) is an endogenously produced gas that is toxic at high concentrations. It is eliminated by a dedicated mitochondrial sulfide oxidation pathway, which connects to the electron transfer chain at the level of complex III. Direct reduction of cytochrome c (Cyt C) by H 2 S has been reported previously but not characterized. In this study, we demonstrate that reduction of ferric Cyt C by H 2 S exhibits hysteretic behavior, which suggests the involvement of reactive sulfur species in the reduction process and is consistent with a reaction stoichiometry of 1.5 mol of Cyt C reduced/mol of H 2 S oxidized. H 2 S increases O 2 consumption by human cells (HT29 and HepG2) treated with the complex III inhibitor antimycin A, which is consistent with the entry of sulfide-derived electrons at the level of complex IV. Cyt C-dependent H 2 S oxidation stimulated protein persulfidation in vitro, while silencing of Cyt C expression decreased mitochondrial protein persulfidation in a cell culture. Cyt C released during apoptosis was correlated with persulfidation of procaspase 9 and with loss of its activity. These results reveal a potential role for the electron transfer chain in general, and Cyt C in particular, for potentiating sulfide-based signaling.

Hydrogen sulfide (H 2 S) is an important biological mediator, and synthetic H 2 S donating molecules provide an important class of investigative tools for H 2 S research. Here, we report esterase-activated H 2 S donors that function by first releasing carbonyl sulfide (COS), which is rapidly converted to H 2 S by the ubiquitous enzyme carbonic anhydrase (CA). We report the synthesis, self-immolative decomposition, and H 2 S release profiles of the developed scaffolds. In addition, the developed esterase-triggered COS/H 2 S donors exhibit higher levels of cytotoxicity than equivalent levels of Na 2 S or the common H 2 S donors GYY4137 and AP39. Using cellular bioenergetics measurements, we establish that the developed donors reduce cellular respiration and ATP synthesis in BEAS 2B human lung epithelial cells, which is consistent with COS/H 2 S inhibition of cytochrome c oxidase in the mitochondrial respiratory chain although not observed with common H 2 S donors at the same concentrations. Taken together, these results may suggest that COS functions differently than H 2 S in certain biological contexts or that the developed donors are more efficient at delivering H 2 S than other common H 2 S-releasing motifs.