title: Redox potential and equilibria in the reductive half-reaction of Vibrio harveyi NADPH-FMN oxidoreductase.
abstract: Vibrio harveyi NADPH:FMN oxidoreductase P (FRP(Vh)) is a homodimeric enzyme having a bound FMN per enzyme monomer. The bound FMN functions as a cofactor of FRP(Vh) in transferring reducing equivalents from NADPH to a flavin substrate in the absence of V. harveyi luciferase but as a substrate for FRP(Vh) in the luciferase-coupled bioluminescent reaction. As part of an integral plan to elucidate the regulation of functional coupling between FRP(Vh) and luciferase, this study was carried out to characterize the equilibrium bindings, reductive potential, and the reversibility of the reduction of the bound FMN in the reductive half-reaction of FRP(Vh). Results indicate that, in addition to NADPH binding, NADP(+) also bound to FRP(Vh) in either the oxidized (K(d) 180 microM) or reduced (K(d) 230 microM) form. By titrations with NADP(+) and NADPH and by an isotope exchange experiment, the reduction of the bound FMN by NADPH was found to be readily reversible (K(eq) = 0.8). Hence, the reduction of FRP(Vh)-bound FMN is not the committed step in coupling the NADPH oxidation to bioluminescence. To our knowledge, such an aspect of flavin reductase catalysis has only been clearly established for FRP(Vh). Although the reductive potentials and some other properties of a R203A variant of FRP(Vh) and an NADH/NADPH-utilizing flavin reductase from Vibrio fischeri are quite similar to that of the wild-type FRP(Vh), the reversal of the reduction of bound FMN was not detected for either of these two enzymes.
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
authors.forename: Benfang
authors.initials: B
authors.lastname: Lei
authors.forename: He
authors.initials: H
authors.lastname: Wang
authors.forename: Yimin
authors.initials: Y
authors.lastname: Yu
authors.forename: Shiao-Chun
authors.initials: SC
authors.lastname: Tu
mesh_terms: D038181:FMN Reductase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009249:NADP / Q000138:chemical synthesis / Q000378:metabolism; D010084:Oxidation-Reduction; D013816:Thermodynamics; D014733:Vibrio / Q000201:enzymology*
chemical_list: D009249:NADP; D038181:FMN Reductase