LEAPS-MPS: Specificity and Mechanism of Azoreductases Versus Nonenzymatic Hydrogen Sulfide Reduction of Azo Compounds Due to the Human Gut Microbiome

In this project, funded by the MPS-LEAPS (Launching Early-Career Academic Pathways) Program and The Chemistry of Life Processes (CLP) program in the Division of Chemistry (CHE) and managed by the Division of Chemistry (CHE), Professor Stack and his students at Providence College will study the chemical breakdown of azo-containing small molecules in the human gut. The trillions of microbes in the human gut can metabolize these compounds affecting their activity. However, the ongoing challenge is identifying which organisms and proteins are responsible and how quickly these transformations occur. Professor Stack and his students will examine the chemical requirements for bacterial enzymes and the hydrogen sulfide produced by bacteria to break the nitrogen-nitrogen double bond found in some drugs (such as sulfasalazine and phenazopyridine) and food dyes (like Red 40 and Yellow 4). This fundamental research could have potential broader impacts in future design of bioactive compounds with consideration of the influence of the gut microbiome. Additionally, this project provides undergraduate students with research opportunities through the work described above and by developing a Course-Based Research Experience in General Chemistry at Providence College. These experiences have potential to improve student retention in STEM fields and participation in scientific research. Professor Stack and his students will synthesize various azobenzene derivatives and evaluate their reduction rates by azoreductase enzymes (AzoRs) and hydrogen sulfide, assessing susceptibility to enzymatic and nonenzymatic reduction. AzoRs are proposed to reduce quinone-like tautomers of azobenzenes. These studies examine how electronic properties influence reduction rates and test the proposed AzoR mechanism. Using cyclic voltammetry, UV-visible spectroscopy, 1H and 13C NMR spectrometry, and mass spectrometry, the research will measure reduction potentials and kinetics, with a focus on the importance of quinone-like intermediates and electronic effects at specific sites of azo-bonded molecules. The goal is to identify compounds reducible by hydrogen sulfide, compare reduction rates with enzymatic processes, and relate reduction potentials to limitations in hydrogen sulfide activity. These insights have potential to clarify which reduction sources operate in the human gut, to support an understanding of bacterial metabolism of drugs and food dyes through metagenomic and metatranscriptomic data. This fundamental research will have broader impacts in the development of bioactive compounds and foster future investigations into enzyme specificity, drug metabolism, and metagenomics. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.