Probing the mechanisms of the biological intermolecular transfer of reduced flavin

NAD(P)H-flavin oxidoreductases [flavin reductases (FR)I are a class of enzy mes capable of producing reduced flavin for bacterial bioluminescence and o ther biological processes. Bacterial luciferase utilizes oxygen, reduced FM N (FMNH2) and a long-chain aliphatic aldehyde as substrates for light emiss ion. The Vibrio harveyi luciferase and FRP (for which we have cloned the ge ne 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-lucifera se coupled assay measuring bioluminescence intensity or quantum output. The FRP exhibits a ping-pong kinetic pattern in the single-enzyme assay but ch anges to a sequential pattern in the coupled assay. Furthermore, FMN at >2 x10(-6) mol/L reduced both the light intensity and quantum yield of the cou pled reaction by noncompetitively inhibiting NADPH and competitively inhibi ting luciferase. These results support a scheme in which the luciferase for ms specific complex(es) with FRP. Indeed, such complexes were shown by fluo rescence anisotropy to exist between luciferase and monomeric FRP either in the holo- or apoenzyme form, Furthermore, the reduced flavin cofactor of F RP is transferred directly to luciferase for bioluminescence, whereas the r educed flavin product of FRP is inefficient in supporting the luminescence reaction. The mechanism of reduced flavin transfer is apparently flavin and Ravin reductase specific.