A case of mistaken identity: are reactive oxygen species actually reactive sulfide species?

Abstract

Stepwise one-electron reduction of oxygen to water produces reactive oxygen species (ROS) that are chemically and biochemically similar to reactive sulfide species (RSS) derived from one-electron oxidations of hydrogen sulfide to elemental sulfur. Both ROS and RSS are endogenously generated and signal via protein thiols. Given the similarities between ROS and RSS, we wondered whether extant methods for measuring the former would also detect the latter. Here, we compared ROS to RSS sensitivity of five common ROS methods: redox-sensitive green fluorescent protein (roGFP), 2', 7'-dihydrodichlorofluorescein, MitoSox Red, Amplex Red, and amperometric electrodes. All methods detected RSS and were as, or more, sensitive to RSS than to ROS. roGFP, arguably the "gold standard" for ROS measurement, was more than 200-fold more sensitive to the mixed polysulfide H2Sn(n = 1-8) than to H2O2 These findings suggest that RSS may be far more prevalent in intracellular signaling than previously appreciated and that the contribution of ROS may be overestimated. This conclusion is further supported by the observation that estimated daily sulfur metabolism and ROS production are approximately equal and the fact that both RSS and antioxidant mechanisms have been present since the origin of life, nearly 4 billion years ago, long before the rise in environmental oxygen 600 million years ago. Although ROS are assumed to be the most biologically relevant oxidants, our results question this paradigm. We also anticipate our findings will direct attention toward development of novel and clinically relevant anti-(RSS)-oxidants.

Garlic oil polysulfides: H2S- and O2-independent prooxidants in buffer and antioxidants in cells

“…7). This is similar to our previous report (8) in which 1 M of the mixed polysulfide, H 2 S n , also effectively oxidized roGFP. Furthermore, while cysteine and glutathione reduced roGFP, these effects could be overcome by garlic oil oxidation at substantially lower concentrations of garlic oil.…”

“…Conversely, high concentrations (ϳ1-3 mM) of garlic oil were able to reduce the oxidized roGFP (Fig. 8), which was also similar to the effects of H 2 S n at these same two high concentrations (8).…”

“…The issue is further complicated by the possibility of tautomerization between thiosulfoxides and polysulfides (55). We have shown previously (8) and again in Fig. 2 that SSP4 readily responds to dihydropersulfides and dihydropolysulfides; however, the decrease in fluorescence at high H 2 S n or garlic oil concentrations (see below) may reflect this persulfide-SSP4 interaction.…”

“…Both prooxidant and antioxidant activities have been ascribed to H 2 S and related polysulfides (H 2 S n ; n ϭ 2-8), although there is increasing evidence that their prooxidant effects are considerably more prevalent than originally thought (8,40). Arguably, the greatest interest in H 2 S and polysulfide signaling is in their ability to bind to a protein cysteine through a process called sulfhydration, or more appropriately persulfidation (11), and, thereby, change the functional properties of that protein (11,19,20,37,44).…”

“…We (8) recently observed that H 2 S and related polysulfides readily oxidize a number of indicators commonly used to detect redox state and reactive oxygen species, including redox-sensitive green fluorescent protein (roGFP), 2=,7=-dichlorofluorescein (DCF), MitoSox Red, and Amplex Red. In the present studies, we used two of these indicators, roGFP and DCF, along with specific fluorescent indicators of H 2 S (7-azido-4-methylcoumarin, AzMC) and 3=,6=-Di(O-thiosalicyl) fluorescein polysulfides (SSP4) to examine H 2 S and polysulfides release from garlic oil and DATS and to determine their potential to act as an oxidant or reductant.…”