Sulfide-regulated copper metabolism - Hydrogen sulfide (H2S) is a signaling molecule that regulates energy metabolism and influences a range of physiological processes in the cardiovascular, central nervous and gastrointestinal systems. Low H2S stimulates respiration as a substrate for the electron transport chain (ETC), supplying electrons at the level of coenzyme Q via sulfide quinone oxidoreductase (SQOR). High H2S inhibits respiration by targeting the copper and heme a3 sites in complex IV in the ETC. While complex IV inhibition has been studied in vitro, the mechanism of its long- term inhibition and metabolic consequences in vivo are unknown. Sustained inhibition of complex IV is especially relevant in intestinal epithelial cells, which are routinely exposed to high H2S (0.2-2.4 mM) derived from gut microbial metabolism. Preliminary data from our laboratory indicate that chronic sulfide exposure elicits profound changes in copper metabolism in colon adenocarcinoma cells, modulates expression of copper-binding proteins, and increases whole cell and mitochondrial copper concentrations. Furthermore, chronic sulfide exposure leads to a marked reduction in the copper-containing complex IV subunits, MT-CO1 and MT-CO2, but does not affect cell viability. These studies reveal a previously unknown intersection between sulfide and copper homeostasis and inform the hypothesis that sulfide leaches copper from complex IV subunits, destabilizes MT-CO1 and MT- CO2, and triggers reorganization of subcellular copper stores, leading to long-term inhibition of respiration and metabolic reprogramming. I will test this hypothesis by addressing the following specific aims: (i) I will characterize sulfide-responsive copper metabolism across a range of primary and transformed cell lines cultivated in a growth chamber with 25-500 ppm H2S in the atmosphere and measure whole cell and organellar copper levels as well as complex IV protein levels. I will monitor copper distribution via X-ray fluorescence microscopy and localize copper transporters by immunofluorescence microscopy, respectively. I will assess how sulfide clearance influences copper metabolism in SQOR-deficient intestinal cells as well as in colon derived from Villin-Cre SQORfl/fl mice. (ii) I will evaluate whether complex IV poses a unique vulnerability among cuproenzymes to chronic sulfide exposure by determining whether protein levels and activities of other cuproenzymes (e.g., superoxide dismutase and lysyl oxidase) are impacted by sulfide exposure in cell lines and in intestinal tissue from Villin-Cre SQORfl/fl mice. I will compare the effects of sulfide with other ETC inhibitors (e.g., cyanide and antimycin A). Finally, copper oxidation and sulfur coordination states will be analyzed by X- ray absorption spectrometry, to determine whether insoluble copper sulfide is formed upon chronic sulfide exposure. The successful completion of these aims will provide fundamentally new insights into the regulation of energy metabolism via sulfide-mediated changes in copper metabolism and simultaneously provide me with critical research training and career development opportunities.
