NAD(P)H:quinone oxidoreductases (NQOs) are flavin-dependent enzymes that catalyze the two-electron reduction of quinones and the oxidation of NAD(P)H. NQOs play a role in cellular detoxification by preventing the formation of radical quinones thus avoiding the generation of reactive oxygen species. The NQO from Pseudomonas aeruginosa PAO1 (PA1024) is proposed to serve a dual function in the cell by detoxifying quinones and balancing the [NAD⁺]/[NADH] ratio. Crystal structures of wild-type PA1024 were previously solved in free form and in complex with NAD⁺ (PDB: 2GJN & 6E2A). Comparison of the ligand-free and ligand-bound structures of the enzyme reveals a loop (residues 75-86) in two conformations, suggesting the mobile loop may play a role in NADH binding and selectivity. P78 is located on the mobile loop and is hypothesized to provide internal rigidity to the mobile loop to help carry out its functions. In the present study, site-directed mutagenesis was utilized to replace P78 with glycine and increase mobile loop flexibility. The mutant (NQO-P78G) was expressed and purified as previously described for wild-type PA1024. Apparent steady-state kinetics of NQO-P78G, at varying concentrations of benzoquinone and a fixed [NADH] of 0.1 mM, showed a 20-fold reduction in the k_cat value with respect to the wild-type enzyme, i.e., 1.3 s^-1 vs. 27 s^-1. When the concentration of NADH was varied with a fixed [benzoquinone] at 21 μM, NQO-P78G could not be saturated and only a k_cat/K_NADH value of 11,000 ± 700 M^-1 s^-1 could be determined. On the basis of kinetic results presented here, we propose that P78 does not play a role in substrate specificity but rather it is important in assisting with catalysis in PA1024.