Complex formation between Vibrio harveyi luciferase and monomeric NADPH:FMN oxidoreductase.
A direct transfer of the reduced flavin mononucleotide (FMNH(2)) cofactor of Vibrio harveyi NADPH:FMN oxidoreductase (FRP) to luciferase for the coupled bioluminescence reaction has been indicated by recent kinetic studies [Lei, B., and Tu, S.-C. (1998) Biochemistry 37, 14623-14629; Jeffers, C., and Tu, S.-C. (2001) Biochemistry 40, 1749-1754]. For such a mechanism, a complex formation of luciferase with FRP is essential, but until now, no evidence for such a complex has been reported. In this work, FRP was labeled at 1:1 molar ratio with the fluorophore eosin. The labeled enzyme was about 30% active in either the reductase single-enzyme or the luciferase-coupled assay. The labeled FRP in either the holo- or apoenzyme form was similar to the native FRP in undergoing a monomer-dimer equilibrium. By measuring the steady-state fluorescence anisotropy of eosin-labeled FRP, it was shown that luciferase formed a complex at 1:1 molar ratio with the monomer of either the apoenzyme or the holoenzyme form of FRP with K(d) values of 7 and 11 microM, respectively. Neither the holo- nor the apoenzyme of the labeled FRP in the dimeric form was effective in complexing with luciferase. At maximal in vivo bioluminescence, the V. harveyi cellular contents of luciferase and FRP were estimated to be 172 and 3 microM, respectively. The vast majority of FRP would be trapped in the luciferase/FRP complex. Plausible physiological significance of such a finding is discussed.
Jeffers
Christopher E
CE
Department of Biology, University of Houston, Houston, Texas 77204, USA.
Nichols
Jeffry C
JC
Tu
Shiao-Chun
SC
D019281:Dimerization; D038181:FMN Reductase / Q000737:chemistry* / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism; D005454:Fluorescence Polarization; D007424:Intracellular Fluid / Q000201:enzymology; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D014461:Ultracentrifugation; D014733:Vibrio / Q000201:enzymology*
D046911:Macromolecular Substances; D009249:NADP; D005486:Flavin Mononucleotide; D008156:Luciferases; D038181:FMN Reductase