Arginine substitution of conserved Lys609 and Lys733 impairs FMN dynamics, electron transfer, and nitric oxide production in endothelial nitric oxide synthase.

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO), a key molecule for maintaining vascular health. While phosphorylation is a well-established regulatory mechanism of eNOS activity, the functional contribution of conserved lysine residues to electron transfer and catalytic coupling remains less clearly defined. In this study, we examined two conserved lysines in eNOS, Lys609 located within the autoinhibitory (AI) region and Lys733 positioned within the FMN-FNR hinge, by substituting them with arginine to preserve positive charge while altering side-chain geometry. Biochemical and spectroscopic analysis revealed that both substitutions significantly impaired enzyme function. Cytochrome c reductase activity was reduced by 3-6-fold, and NO synthesis decreased by approximately 37% for K609R and 25% for K733R relative to wild-type (WT) eNOS. Elevated NADPH/NO ratios indicated impaired catalytic coupling and increased diversion of electrons away from productive NO synthesis. Flavin fluorescence and auto-oxidation measurements showed that both mutations favored a closed, FMN-shielded conformation and reduced the Ca2+/calmodulin-induced transition to the open, catalytically competent state. Structural analyses and Molecular Dynamics simulations show that substitutions at Lys609 and Lys733 alter FMN-domain dynamics through distinct mechanisms. K609R induces increased flexibility and global expansion of the reductase domain, and K733R restricts hinge motion, maintaining overall compactness. Despite these defects, ferricyanide reductase activity was unchanged, showing that FAD-mediated hydride transfer remains unaffected. Electron flux through the heme correlated strongly with NO production, identifying heme-directed electron transfer as the principal step affected. Together, these findings suggest Lys609 and Lys733 as regulators of eNOS conformational dynamics, interdomain electron transfer, and catalytic efficiency.