Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1

Enzymes require flexible regions to adopt multiple confor-mations during catalysis. The mobile regions of enzymes include gates that modulate the passage of molecules in and out of the enzyme's active site. The enzyme PA1024 from Pseudomonas aeruginosa PA01 is a recently discovered flavin-dependent NADH:quinone oxidoreductase (NQO, EC 1.6.5.9). Q80 in loop 3 (residues 75-86) of NQO is similar to 15 angstrom away from the flavin and creates a gate that seals the active site through a hydrogen bond with Y261 upon NADH binding. In this study, we mutated Q80 to glycine, leucine, or glutamate to investigate the mechanistic significance of distal residue Q80 in NADH binding in the active site of NQO. The UV-visible absorption spectrum reveals that the mutation of Q80 minimally affects the protein microenvironment surrounding the flavin. The anaerobic reductive half-reaction of the NQO-mutants yields a >= 25-fold increase in the K-d value for NADH compared to the WT enzyme. However, we determined that the k(red) value was similar in the Q80G, Q80L, and wildtype enzymes and only X25% smaller in the Q80E enzyme. Steady-state kinetics with NQO-mutants and NQO-WT at varying concentrations of NADH and 1,4-benzoquinone establish a <= 5-fold decrease in the k(cat)/K-NADH value. Moreover, there is no significant differ-ence in the k(cat)/K-BQ (similar to 1 x 10(6) M(-1)s(-1)) and k(cat) (similar to 24 s(-1)) values in NQO-mutants and NQO-WT. These results are consistent with the distal residue Q80 being mechanistically essential for NADH binding to NQO with minimal effect on the quinone binding to the enzyme and hydride transfer from NADH to flavin.

Automated summary

Enzymes require flexible regions for catalysis, and the mobile regions of enzymes include gates that regulate the passage of molecules in and out of the active site. Q80 in loop 3 of the enzyme NQO plays a mechanistically essential role in NADH binding, but does not significantly affect quinone binding and hydride transfer.