Mechanistic and Molecular Dynamics Studies Reveal that Increased Loop 3 Mobility Alters Substrate Capture in an NADH:Quinone Oxidoreductase

Dynamic protein loops can act as molecular gates that stabilize enzyme-substrate complexes, yet the underlying motions are poorly defined. Here, we dissect the role of loop 3 in an NADH:quinone oxidoreductase (NQO, UniProt Q9I4V0) from <i>Pseudomonas aeruginosa</i> PA01 in governing substrate binding and catalysis. Previous mechanistic and structural studies proposed that loop 3 fluctuations regulate substrate binding; however, an associated atomic-level understanding of the conformational changes is lacking. We probe the role of loop 3 dynamics in substrate capture and catalysis by mutating conserved P78 to glycine, which perturbs the gate rigidity. Steady-state kinetics with NQO-P78G and NQO-WT at varying concentrations of NADH and coenzyme Q₀ established a 3.5-fold decrease in the <i>K</i>_CoQ0 value, a 2.0-fold reduction in the <i>k</i>_cat value, and a 1.8-fold increase in the <i>k</i>_cat/<i>K</i>_CoQ0 value. The anaerobic reductive half-reaction of NQO-P78G with NADH yielded a ≤3.5-fold decrease in the <i>k</i>_red value and an estimated 80-fold increase in the <i>K</i>_d value compared to NQO-WT. Molecular dynamics simulations of ligand-free NQO-P78G and NQO-WT suggest that the P78G mutation disrupts interdomain interactions, allowing loop 3 to sample more open conformations. The combination of mechanistic and computational experiments suggests that more open gate conformations minimally promote access of the smaller coenzyme Q₀ substrate to the active site. In contrast, the bulkier NADH substrate is less likely to associate, as the more open conformations prevent key interactions with NQO gate residues from forming. These results build on previous studies with NQO by demonstrating that altering loop 3 gate rigidity modulates substrate binding.