Probing the mechanisms of the biological intermolecular transfer of reduced flavin

Abstract

NAD(P)H-flavin oxidoreductases [flavin reductases (FR)] are a class of enzymes capable of producing reduced flavin for bacterial bioluminescence and other biological processes. Bacterial luciferase utilizes oxygen, reduced FMN (FMNH2) and a long-chain aliphatic aldehyde as substrates for light emission. The Vibrio harveyi luciferase and FRP (for which we have cloned the gene and determined the crystal structure) is a model for the elucidation of the reduced flavin transfer mechanism using both a flavin reductase single-enzyme assay monitoring the NADPH oxidation and a flavin reductase-luciferase coupled assay measuring bioluminescence intensity or quantum output. The FRP exhibits a ping-pong kinetic pattern in the single-enzyme assay but changes to a sequential pattern in the coupled assay. Furthermore, FMN at >2x10(-6) mol/L reduced both the light intensity and quantum yield of the coupled reaction by noncompetitively inhibiting NADPH and competitively inhibiting luciferase. These results support a scheme in which the luciferase forms specific complex(es) with FRP. Indeed, such complexes were shown by fluorescence anisotropy to exist between luciferase and monomeric FRP either in the holo- or apoenzyme form. Furthermore, the reduced flavin cofactor of FRP is transferred directly to luciferase for bioluminescence, whereas the reduced flavin product of FRP is inefficient in supporting the luminescence reaction. The mechanism of reduced flavin transfer is apparently flavin and flavin reductase specific.