Thermodynamic analysis of the binding of oxidized and reduced FMN cofactor to Vibrio harveyi NADPH-FMN oxidoreductase FRP apoenzyme.
The Vibrio harveyi NADPH-specific flavin reductase FRP follows a ping-pong mechanism but switches to a sequential mechanism in the luciferase-coupled reaction. The bound FMN co-isolated with FRP, while acting as a genuine cofactor in the single-enzyme reaction, functions in the luciferase-coupled reaction as a prebound substrate and is directly transferred to luciferase once it is reduced [Lei, B., and Tu, S.-C. (1998) Biochemistry 37, 14623-14629]. With the aim of better understanding the functions of FMN in the FRP holoenzyme, this study was undertaken to quantify and compare the thermodynamic properties of the binding of oxidized and reduced FMN by the FRP apoenzyme. By isothermal titration calorimetry (ITC) measurements in various buffers at pH 7.0 and 15-30 degrees C, the binding of FMN by apo-FRP was found to be noncooperative, exothermic, and primarily enthalpy driven. The binding free energy change (hence, the association constant) was nearly invariant over this temperature range. Significant conformational changes in FRP upon binding of FMN were indicated. Equilibrium bindings of reduced flavins by flavin-dependent proteins have rarely been studied. In this work, the thermodynamic properties of binding of reduced FMN by apo-FRP were found to closely resemble those of FMN binding under three sets of experimental conditions via ITC measurements and, in one case, fluorescence quenching. The kinetically deduced ping-pong mechanism of FRP is now supported by direct measurements of binding affinities of the oxidized and reduced FMN cofactors. These findings are also discussed in relation to the function of FRP as a reduced flavin donor in the FRP-luciferase couple.
Li
Xi
X
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
Chow
Dar-Chone
DC
Tu
Shiao-Chun
SC
D001051:Apoenzymes / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D038181:FMN Reductase / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014733:Vibrio / Q000201:enzymology*
D001051:Apoenzymes; D001426:Bacterial Proteins; D005486:Flavin Mononucleotide; D038181:FMN Reductase