Flavin specificity and subunit interaction of Vibrio fischeri general NAD(P)H-flavin oxidoreductase FRG/FRase I

Apoenzyme of the major NAD(P)H-utilizing flavin reductase FRG/FRase I from Vibrio fischeri was prepared. The apoenzyme bound one FMN cofactor per enzy me monomer to yield fully active holoenzyme. The FMN cofactor binding resul ted in substantial quenching of both the flavin and the protein fluorescenc e intensities without any significant shifts in the mission peaks. In addit ion to FMN binding (K-d 0.5 muM at 23 degreesC), the apoenzyme also bound 2 -thioFMN, FAD and riboflavin as a cofactor with K-d values of 1, 12, and 37 muM, respectively, at 23 degreesC. The 2-thioFMN containing holoenzyme was about 40% active in specific activity as compared to the FMN-containing ho loenzyme. The FAD- and riboflavin-reconstituted holoenzymes were also catal ytically active but their specific activities were not determined. FRG/FRas e I followed a ping-pong kinetic mechanism. It is proposed that the enzyme- bound FMN cofactor shuttles between the oxidized and the reduced form durin g catalysis. For both the FMN- and 2-thioFMN-containing holoenzymes, 2-thio FMN was about 30% active as compared to FMN as a substrate. FAD and ribofla vin were also active substrates. FRG/FRase I was shown by ultracentrifugati on at 4 degreesC to undergo a monomer-dimer equilibrium, with K-d values of 18.0 and 13.4 muM for the apo- and holoenzymes, respectively. All the spec tral, ligand equilibrium binding, and kinetic properties described above ar e most likely associated with the monomeric species of FRG/FRase 1. Many as pects of these properties are compared with a structurally and functionally , related Vibrio harveyi NADPH-specific flavin reductase FRP. (C) 2001 Acad emic Press.