VIBRIO-HARVEYI NADPH-FMN OXIDOREDUCTASE - PREPARATION AND CHARACTERIZATION OF THE APOENZYME AND MONOMER-DIMER EQUILIBRIUM

A rapid chromatography method was developed for the preparation of apo enzyme of Vibrio harveyi NADPH:FMN oxidoreductase with greater than or equal to 80% yields. The apoenzyme bound one FMN per enzyme monomer w ith a dissociation constant of 0.2 mu M at 23 degrees C. The reconstit uted holoenzyme was catalytically as active as the native enzyme. FMN binding resulted in 87 and 92% of quenching of protein and flavin fluo rescence, respectively, indicating a conformational difference between the apoprotein and the holoenzyme. Neither riboflavin nor FAD showed any appreciable binding to the cofactor site of the apoenzyme but both flavins were active substrates for the FMN-containing holoenzyme. 2-T hioFMN bound to the cofactor site of the apoenzyme with an affinity si milar to that for FMN binding, The holoenzyme reconstituted with 2-thi oFMN showed a 509-nm absorption peak, which represents a 19-nm red shi ft from the corresponding peak of the free flavin, and was catalytical ly active in using either FMN or 2-thioFMN as a substrate, The holoenz yme showed a concentration dependence in molecular sieve chromatograph y corresponding to higher apparent molecular weights at higher concent rations. Both the holoenzyme and the apoenzyme was shown at 4 degrees C by equilibrium ultracentrifugation to undergo dimerization with diss ociation constants of 1.8 and 3.3 mu M, respectively. (C) 1997 Academi c Press, Inc.