The liminal zone between host epithelium and gut microbiota is rich in sulfide conversations that are modulated by diet; their short- and long-range impacts on systemic H2S homeostasis represent a largely unexplored terrain ripe for discovery. The toxicity of H2S, first described as a noxious “acidic vapor” some 500 years ago, results from its inhibition of complex IV in the electron transport chain (ETC) and explains the ubiquitous presence of sulfide quinone oxidoreductase (SQOR) in mitochondria along with other sulfide oxidation pathway enzymes, for H2S disposal. The past cycle witnessed exciting advances, including the discovery of an unorthodox redox cofactor configuration, comprising an active site trisulfide, which affords the SQOR reaction an ~105-fold catalytic advantage, and characterization of the first clinical mutations in SQOR, which present with brain pathology typical of Leigh disease. We discovered that plasticity embedded in the mammalian ETC, which allows diversion of electrons to fumarate as a terminal acceptor when the capacity to use O2 is restricted by H2S. The ensuing reductive shift in the ETC, propagates beyond the mitochondrion via metabolic shuttles and redox cofactor pools, enhancing aerobic glycolysis, lipid biogenesis and reductive carboxylation in the Krebs cycle. In the next cycle, we will build on this exciting momentum as well as a wealth of preliminary data, introducing multi- omics approaches (metabolomics, RNA-Seq and CRISPRi) combined with cellular and genetic models of inducible SQOR deficiency (whole body or intestinal epithelium) to the rigorous biochemical foundations of our program. We will characterize the allosteric regulation of H2S biogenesis and clearance by metabolites identified from an unbiased screen, quantify the potential of H2S to tune O2 affinity of complex IV, and assess its impact on the hypoxic response. We will focus on the host-gut microbe interface, which we estimate accounts for ~70% of systemic sulfide homeostasis, and evaluate how its modulation by dietary methionine, microbial composition and host genotype influences brain pathology in control versus SQOR deficient mice. Advanced microscopic techniques, including live-cell imaging with biosensors to monitor ATP pools and redox shifts, will be used to elucidate H2S-induced changes to mitochondrial membrane potential, morphology and fusion. And we will uncover the molecular basis of the correlation between a graded decrease in gut reuterin (derived from Lactobacillus reuteri), and increasing tumor burden in colorectal cancer and the associated increase in host sulfur metabolites. We will continue to leverage the flexibility of the R35 funding mechanism for the sustained and innovative interrogation of the impact of H2S chemical biology in health and disease.