Glutamine Substitution at Lysine ⁶⁰⁹ in the Autoinhibitory Region Modulates Catalysis, Uncoupling, and Conformational Dynamics of Endothelial Nitric Oxide Synthase

Endothelial nitric oxide synthase (eNOS) is a critically important flavoheme enzyme that synthesizes nitric oxide (NO), a multifunctional signaling molecule essential to vascular homeostasis, immune modulation, and cardiovascular function. Post-translational modifications, such as acetylation, can affect eNOS activity by altering the charge state of lysine residues. However, we presently have little knowledge of how each lysine contributes to catalysis. In this study, we biochemically characterize two conserved lysine residues (Lys⁶⁰⁹ and Lys⁷³³) of eNOS by employing lysine-to-glutamine substitutions (K609Q and K733Q), which neutralize the positive charge at these sites. The K609Q mutant showed an 80% increase in NO production and a better coupling efficiency (∼2.9 vs ∼5.2 in wild-type). This suggests that both electron transfer and enzymatic uncoupling were more effective. This mutant exhibited 1.5-fold enhanced cytochrome <i>c</i> reductase activity and accelerated flavin autoxidation, indicating an increased electron flux through the flavoprotein domain and decreased FMN semiquinone stability. Flavin fluorescence analysis further revealed that K609Q favors a partially open conformational equilibrium that supports enhanced interdomain electron transfer. Ferricyanide reduction remained unchanged across all variants, confirming that FAD-dependent electron transfer is unaffected and that the K609Q substitution specifically modulates FMN-mediated, not FAD-linked, electron flux. Conversely, K733Q exhibited wild-type-like behavior with little to no effect on coupling efficiency and catalysis. Our findings reveal that Lys⁶⁰⁹ serves a regulatory function in eNOS catalysis. Substitution of this residue enhances coupling efficiency and promotes improved electron transfer. These insights advance our understanding of eNOS enzymology and indicate that the modulation of specific lysine residues, particularly Lys⁶⁰⁹, may aid in the preservation of endothelial NO production and alleviate the redox imbalance associated with endothelial dysfunction.