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), namely peroxide (H2O2), superoxide (O2.−) and the hydroxyl radical (HO.−). These are chemically and biochemically similar to reactive sulfide species (RSS) that are derived from one‐electron oxidation of hydrogen sulfide (H2S) to elemental sulfur, namely persulfide (H2S2), “supersulfide” (S2.−) and the thiyl radical (HS.−). H2S is also reactive and can act as a RSS. Both ROS and RSS are endogenously generated, signal via reactive protein thiols and have common effector targets. Given these similarities, we hypothesized that extant methods for measuring the former would also detect the latter. Here we compared the sensitivity of five methods commonly employed for measuring ROS to both ROS (H2O2 and superoxide as KO2), and to RSS (H2S, H2Sn where n=2–8, H2S2, H2S3 and H2S4. The methods examined were redox‐sensitive green fluorescent protein (roGFP), 2′, 7′‐dihydrodichlorofluorescein (DCF), MitoSox Red, Amplex Red and H2O2 amperometric electrodes. All methods detected RSS and were as or more sensitive to RSS than they were to ROS. roGFP, arguably the “gold standard” for ROS measurement, was over two‐hundred fold more sensitive to H2Sn (n=2–8) than H2O2. Likewise, ROS and RSS produced dose‐dependent increases in 10 ìM DCF fluorescence, with high (1, 3 mM) H2S giving the most rapid increase in DCF fluorescence. H2O2 and H2S were essentially equipotent, whereas KO2 was considerably less efficacious. Lastly, amperometric H2O2 sensors were nearly 30 times more sensitive to H2S than to H2O2 and two or three times more sensitive to H2S2, H2S3 and H2S4 than to H2O2. The responses of roGFP and DCF to RSS were not diminished by hypoxia (Po2 <4 mmHg) which indicates that they did not involve ROS generation from RSS. The estimated daily production of RSS by cysteine catabolism is similar to estimated ROS production from oxygen consumption, but may exceed it if ROS production is overestimated due to RSS interference. These findings suggest that RSS are 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 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.Support or Funding InformationNSF IOS‐144‐6310 (KRO), NSF GRFP DGE‐131‐3583 (ERD)