Efficient inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by sulfuration with solubilized elemental sulfur

Hydrogen sulfide (H2S), carbon monoxide (CO), and nitric oxide (NO) have garnered increasing scientific interest in recent decades due to their classifications as members of the gasotransmitter family of signaling molecules. Due to the versatility of sulfur redox chemistry in biological systems, H2S specifically is being studied for its ability to modulate cellular redox environments, particularly through the downstream production of oxidized sulfur species. A major mechanism of this regulation is through a posttranslational modification known as persulfidation, where oxidized sulfur atoms are appended to free cysteine in proteins. Currently, it is difficult to discern the activity of H2S itself versus these oxidized sulfur species, particularly sulfane sulfur (S0). We have previously developed a method of solvating S8, a source of pure S-0, to more accurately study persulfidation and sulfuration in general. Here, we apply this pure S-0 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has previously been shown to be inhibited by S-0-containing polysulfides via persulfidation. Using solvated S0, we demonstrate that native, reduced GAPDH can be completely inhibited by sulfuration with S-0. Further, oxidized GAPDH activity cannot be rescued using S-0, demonstrating that it is the oxidation of reduced GAPDH by S-0 that curtails its activity. We also compare inhibition of GAPDH by pure S-0 to different polysulfides and demonstrate the modulating effects that pendant alkyl groups have on GAPDH inhibition. These results highlight the promise of this novel, simplified system for the study of S-0.