Hydrogen Sulfide and Other Reactive Sulfide Species’ Role in Cellular Signaling and Oxygen Sensing

Hydrogen sulfide (H₂S) and other reactive sulfide species (RSS) were thought of as purely toxic chemicals 20 years ago, but since have been implicated in many important cellular signaling pathways with numerous physiological functions. In this thesis, these functions are examined in depth, including reaction with and mismeasurement of reactive oxygen species (ROS) for RSS and whether RSS are acting as, or at least implicated in, endogenous oxygen sensing. RSS and ROS measurement via amperometric sensors are first shown to have great cross-sensitivity, particularly ROS sensors for RSS in solution, which is shown to be up to 100-fold greater response for RSS than ROS. Next, newly introduced fluorescent molecules designed to be specific to particular reactive oxygen species and reactive sulfide species were analyzed, showing unequivocally that ROS-specific fluorophores are as sensitive or many times more sensitive to RSS than to their target molecules. Given this cross-sensitivity, we next investigated the possible chemical reactions of RSS and the ways they may be produced in cells. Garlic proved to be a potent releaser of H₂S given that small, reduced thiols were present, and also releases polysulfides naturally. Next, we looked into ROS enzymes, as their original function may have instead dealt with RSS. Catalase was found to generate H₂S from certain RSS, showing that catalase can act as a sulfur oxidase or sulfur reductase. Additionally, our experiments will show that superoxide dismutase (SOD) can also catalyze a reaction using H₂S and O₂ to form persulfides, which can then combine in various ways to form polysulfides and sulfur-oxides. Being able to delineate ROS from RSS and understanding the many potential routes for RSS production, the final goal of this research was to show RSS production in vitro, particularly in hypoxia. Our results suggest that H₂S production is greatly increased in hypoxia in two primary cell lines. This is further supported by mRNA levels of H₂S degradation enzymes decreasing in hypoxia and the relative protein abundance of H₂S producing enzymes increasing, providing support for H₂S and RSS’ involvement in oxygen sensing.