Abstract
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 Pseudomonas aeruginosa 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 Q(0) established a 3.5-fold decrease in the K(CoQ0) value, a 2.0-fold reduction in the k(cat) value, and a 1.8-fold increase in the k(cat)/K(CoQ0) value. The anaerobic reductive half-reaction of NQO-P78G with NADH yielded a ≤3.5-fold decrease in the k(red) value and an estimated 80-fold increase in the K(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 Q(0) 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.