Dextromethorphan Is a Novel Pharmacological Inhibitor of F ₁ F O ‐ ATPase That Targets the Membrane‐Embedded Domain Impairing ATP Synthesis and Hydrolysis

Dextromethorphan (DXM), a widely used antitussive agent, was investigated for its effects on mitochondrial F 1 F O ‐ATPase activity and oxidative phosphorylation. Our results demonstrate that DXM inhibited F 1 F O ‐ATPase independently of the thiol redox state. Mutual exclusion analysis highlighted an overlapping binding site between DXM and dicyclohexylcarbodiimide (DCCD), indicating a shared or adjacent binding site in the membrane‐embedded F O domain of the enzyme. These findings suggested that DXM selectively targeted the proton translocation mechanism of F 1 F O ‐ATPase during the ATP hydrolysis and synthesis of ATP. Moreover, kinetic analysis confirmed a high affinity of DXM for the enzyme, with an inhibitory efficiency of 2.37 mM −1 ⸱s −1 . Importantly, DXM did not affect electron transport chain activity but impaired ATP synthesis, as evidenced by altered respiratory control ratios of oxidative phosphorylation. The data obtained offer new insights into its off‐target mitochondrial effects and potential implications for bioenergetic regulation.