ABSTRACT NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing reactive oxygen species. Since coenzyme Q (CoQ) mimics affect NOX4 activity, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, modulates inflammatory activation. Mice lacking endothelial CYB5R3 (R3 KO), under lipopolysaccharides (LPS) challenge, showed exacerbated hypotension, decreased acetylcholine-induced vasodilation, and elevated vascular adhesion molecule 1 (Vcam-1) mRNA in aorta. In vitro, silencing Cyb5r3 enhanced LPS-induced VCAM-1 protein in a NOX4 dependent manner. APEX2- based electron microscopy and proximity biotinylation demonstrated CYB5R3’s localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 silenced HAECs had less total H 2 O 2 but more mitochondrial O 2 •- . Using inactive or non-membrane bound active CYB5R3, we found CYB5R3 activity and membrane translocation were needed for optimal generation of H 2 O 2 by NOX4. Lastly, CoQ deficient cells showed decreased NOX4-derived H 2 O 2 , indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H 2 O 2 generation via CoQ. NOVELTY AND SIGNIFICANCE What Is Known? NADPH oxidase 4 (NOX4) reportedly produces primarily hydrogen peroxide (H 2 O 2 ) and, to a lesser extent, superoxide (O 2 •- ) and has been shown to have both beneficial and deleterious effects in the cardiovascular system. NOX4 activity can be affected by NAD(P)H quinone oxidoreductase 1 (NQO1), a CoQ reductase, and synthetic quinone compounds used to mimic CoQ. Cytochrome b5 reductase 3 (CYB5R3) is known to reduce CoQ and is highly expressed in endothelial cells. What New Information Does This Article Contribute? In vivo, the lack of endothelial CYB5R3 causes exacerbated lipopolysaccharides (LPS)-induced inflammatory signaling, endothelial dysfunction, and hypotension. Endothelial CYB5R3 mitigates inflammatory signaling by LPS and tumor necrosis factor α (TNF-α) in a NOX4 dependent manner. In endothelial cells, CYB5R3 and NOX4 reside in close proximity on the mitochondrial outer membrane. NOX4’s ability to generate H 2 O 2 depends on the membrane translocation and activity of CYB5R3 and the presence of endogenous CoQ. NONSTANDARD Abbreviations and Acronyms Protein names are abbreviated as capital letters (e.g., CYB5R3), while the corresponding gene names are annotated as in italic lower cases (e.g., Cyb5r3 ).

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation