NADH:quinone oxidoreductase from Pseudomonas aeruginosa PAO1 (NQO, PA1024, EC 1.6.5.9) is a recently reclassified flavin-dependent enzyme that catalyzes the two-electron reduction of a wide variety of quinones to hydroquinone using NADH as an electron donor. The two-electron reduction of quinone plays a detoxification role in P. aeruginosa by avoiding the formation of semiquinone radicals. The previously solved crystal structure of the NQO demonstrated that the substrate-binding site of NQO is formed by a small entrance consisting of a flexible βα loop 3 (residue 75-86). Q80 in loop 3 switches between an open conformation without NAD⁺ bound and a close conformation with NAD⁺ bound. In this study, Q80 was mutated to glycine, leucine, or glutamate through site-directed mutagenesis to investigate the role of Q80 in binding and catalysis in NQO. The results showed that Q80 residue participates in substrate NADH binding in the active site of NQO.