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

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.