Kinetic solvent viscosity effects uncover an internal isomerization of the enzyme-substrate complex in Pseudomonas aeruginosa PAO1 NADH:Quinone oxidoreductase

Abstract

NAD(P)H:quinone oxidoreductases (NQOs) play an essential protective role as antioxidants in the detoxification of quinones in both Prokaryotes and Eukaryotes. NQO from Pseudomonas aeruginosa PAO1 uses FMN to catalyze the two-electron reduction of various quinones with NADH. In this study, steady-state kinetics, kinetic solvent viscosity effects, and rapid reaction kinetics were used to determine which kinetic steps control the overall turnover of the enzyme with benzoquinone or juglone. The rate constant for flavin reduction (kred) at pH 6.0 was 12.9 ± 0.3 s−1, and the Kd for NADH was at least an order of magnitude lower than 90 μM. With benzoquinone, the kcat value was 11.7 ± 0.3 s−1, consistent with flavin reduction being almost entirely rate-limiting for overall turnover. With juglone, a kcat value of 10.0 ± 0.5 s−1 was recorded. The normalized plot of the relative solvent viscosity effects on the kcat values established that hydride transfer from NADH to the FMN and quinol product release, with a calculated rate constant (kP-rel) of 52 s−1, are partially rate-limiting for the overall turnover of NQO. Kinetic solvent viscosity effects with glucose or sucrose revealed a hyperbolic dependence on the kcat and kcat/Km values with benzoquinone or juglone, respectively, consistent with the presence of a solvent-sensitive internal isomerization of the enzyme-substrate complex (ES). The data demonstrate opposing effects of benzoquinone and juglone on the equilibrium of the NQO ES isomerization with glucose or sucrose. Thus, our study demonstrates how quinol substrate properties alter the equilibrium of NQO ES isomerization.