ABSTRACT Our research program seeks to elucidate the chemistry, biology and translational impact of “redox switches” in the cardiovascular system. Our work has revealed that cytochrome b5 reductase 3 (CYB5R3) operates as a critical redox switch in the cardiovascular system by reducing substrates such heme and coenzyme Q to modulate reactive nitrogen and oxygen species and downstream signaling. The overarching goal of this R35 research proposal is to fill four major gaps in our knowledge related to redox switches in the cardiovascular biology. First, we will define the role(s) of the “other CYB5R” family members including CYB5R1, 2, 4, 5 and their functions in endothelial, smooth muscle and cardiomyocytes. While expressed in these cells types, there are no reports defining the function(s) of these enzymes in the cardiovascular system. Using new cell type specific knockouts of CYB5R1, 2, 4, 5, we will delineate the physiological and pathophysiological role(s) of these enzymes across endothelial, smooth muscle and cardiomyocytes and their potential function(s) in redox signaling. Second, we aim to establish novel client proteins for the CYB5R family of enzymes using an innovative “biopanning” approach. Utilizing a genetically modified soybean peroxidase coupled to CYB5R enzymes we will establish new binding partners, substrates and mechanisms for these reductases. Third, using large human data sets (i.e. n=8500 participants), we will assess if common genetic variants in CYB5R enzymes associates with diseases such as hypertension, sickle cell disease and heart failure. Mechanistic studies will determine functional impact of these variants on redox signaling pathways in cardiovascular cells. Forth, we will leverage the information gained from our studies on CYB5R3 to test if a new drug we developed called “NitroQ” and test if improves redox equilibrium and reverses hypertension, sickle cell disease and heart failure. Mechanistic studies will elucidate key targets and downstream signaling pathways influenced by NitroQ. Collectively, these fundamental basic, translational and therapeutic studies will provide a critical framework that will enable us to fill these major gaps in our knowledge and improve our understanding of redox switches in cardiovascular health and disease.