EAGER: Reactive Sulfide Species: Ubiquitous and Primordial Signaling Molecules

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Reactive oxygen species (ROS), superoxide and hydrogen peroxide (H2O2) have long been associated with oxidative stress, intracellular signaling, and deleterious effects in biological systems. However, a large part of the evolution that led to life as we know it and the conservation of mechanisms across species occurred in anoxic and sulfidic environments. Evidence suggests that 'antioxidant pathways' were developed long before the appearance of atmospheric oxygen. Furthermore, many methods for measuring ROS are more sensitive to reactive sulfide species (RSS), which are chemically more reactive and biologically more versatile signaling molecules than ROS. The proposed research examines a novel hypothesis that RSS, hydrogen sulfide (H2S) and polysulfides (H2Sn, n=2-7) are involved in intracellular signaling, and in many instances RSS not ROS are the true endogenous signals and may contribute to oxidative stress. It is also proposed reactive nitrogen species (RNS), nitric oxide (NO-) and nitrite (NO2-) were primordial oxygen acceptors and they interact with RSS in intracellular signaling. This research will offer an alternative to the long-standing and entrenched paradigm that ROS mediate intracellular signaling and oxidative stress. It will show that RSS are highly reactive and versatile signaling molecules in their own right and that many of the well-studied physiological and pathological effects of ROS can be attributed to RSS. The research could open up new fields of intracellular signaling and redox homeostasis, many of which will be applicable in across biology disciplines and could even have considerable therapeutic value. The proposed research will: examine the redox chemistry of RSS; determine the contribution of RSS to redox signaling in cells; and test whether H2S2 may be mistaken for H2O2 by some analytical procedures. Aim 1 will use roGFP to monitor redox activity of RSS and how this is affected by free metals, antioxidant enzymes (e.g., superoxide dismutase, catalase, etc) and heme proteins (hemin, hemoglobin, myoglobin). These studies will be done in normoxia, hypoxia and physiological pO2's, over a range of physiological pH, and in the presence and absence of primordial electron acceptors RNS. Sensitive H2S, O2 and NO amperometric electrodes will be employed to monitor specific RSS reactions and reactions between ROS, RSS and RNS to establish stoichiometric relationships. Aim 2 will examine the specific roles enzymes associated with RSS biosynthesis by inhibition with competitive inhibitors and siRNA. Aim 2 will also examine the effects of exogenous RSS and mitochondrial-targeted H2S releasing drugs on cellular compartmental redox state. Aim 3 will test whether 'specific' ROS assays can distinguish between ROS and RSS. The PI's laboratory has long supported high school, undergraduate, and graduate training and has especially fostered a learning environment for minorities and under-represented groups. This project will provide the resources for this training to continue. Results from the studies will be distributed through presentations at scientific meetings and through peer-reviewed journal publications.