MECHANISM OF REDUCED FLAVIN TRANSFER FROM VIBRIO-HARVEYI NADPH-FMN OXIDOREDUCTASE TO LUCIFERASE

The mechanisms of reduced flavin transfer in biological systems are po orly understood at the present. The Vibrio harveyi NADPH-FMN oxidoredu ctase (FRP) and the luciferase pair were chosen as a model for the del ineation of the reduced flavin transfer mechanism. FRP, which uses FMN as a cofactor to mediate the reduction of the flavin substrate by NAD PH, exhibited a ping-pong kinetic pattern with a K-m,K-FMN of 8 mu M a nd a K-m,K-NADPH Of 20 mu M in a single-enzyme spectrophotometric assa y monitoring the NADPH oxidation. However, the kinetic mechanism of FR P was changed to a sequential pattern with a K-m,K-FMN Of 0.3 mu M and a K-m,K-NADPH of 0.02 mu M in a luciferase-coupled assay measuring li ght emission. In contrast, the Photobacterium fischeri NAD(P)H-FMN oxi doreductase FRG showed the same ping-pong mechanism in both the single -enzyme spectrophotometric and the luciferase-coupled assays. Moreover , for the FRP, FMN at concentrations over 2 mu M significantly inhibit ed the coupled reaction in both light intensity and quantum yield, and showed apparent noncompetitive and competitive inhibition patterns ag ainst NADPH and luciferase, respectively. No inhibition of the NADPH o xidation was detected under identical conditions. These results are co nsistent with a scheme that the reduced flavin cofactor of FRP is pref erentially utilized by luciferase for Light emission, the reduced flav in product generated by the reductase is primarily channeled into a da rk oxidation, and luciferase competes against flavin substrate in reac ting with the FRP reduced flavin cofactor. An FRP derivative containin g 2-thioFMN as the cofactor was also used to further examine the mecha nism of flavin transfer. Results again indicate a preferential utiliza tion of the reductase reduced flavin cofactor by luciferase for the bi oluminescence reaction.