Identity of the emitter in the bacterial luciferase luminescence reaction: binding and fluorescence quantum yield studies of 5-decyl-4a-hydroxy-4a,5-dihydroriboflavin-5'-phosphate as a model.
The excited state of 4a-hydroxy-4a,5-dihydroFMN has been postulated to be the emitter in the bacterial bioluminescence reaction. However, while the bioluminescence quantum yield of the luciferase emitter is about 0.16, chemiluminescence and fluorescence quantum yields of earlier flavin models mimicking the luciferase emitter were no more than 10(-5). To further examine the proposed chemical identity of the luciferase emitter, 5-decyl-4a-hydroxy-4a,5-dihydroFMN was prepared as a new flavin model. Both the wild-type Vibrio harveyi luciferase and a catalytically active alphaC106A mutant formed complexes with the flavin model at a 1:1 molar ratio with K(d) values at 2.4 and 1.2 microM, respectively. This flavin model inhibited the activity of both luciferases, suggesting that it was bound to the enzyme active center. While the free flavin model was itself only very weakly fluorescent, its binding to either luciferase species resulted in markedly enhanced fluorescence, peaking at 440 nm. The fluorescence quantum yields of 5-decyl-4a-hydroxy-4a,5-dihydroFMN bound to wild-type and alphaC106A luciferases were 0.08 and 0.05, respectively, which are about 50% of the respective emitter bioluminescence quantum yields of these two luciferases. The present findings clearly demonstrated that the luciferase active site was suitable for marked enhancement of fluorescence of 4a-hydroxyflavin and, hence, provides a strong support to the proposed identity of 4a-hydroxy-4a,5-dihydroFMN, in its exited state, as the luciferase emitter.
Lei
Benfang
B
Department of Biology, University of Houston, Houston, Texas 77204-5001, USA.
Ding
Qizhu
Q
Tu
Shiao-Chun
SC
D001665:Binding Sites; D005486:Flavin Mononucleotide / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology; D005453:Fluorescence; D049453:Luciferases, Bacterial / Q000037:antagonists & inhibitors / Q000737:chemistry*; D049408:Luminescent Agents / Q000737:chemistry*; D008956:Models, Chemical
C495979:5-decyl-4a-hydroxy-4a,5-dihydroriboflavin-5'-phosphate; D049408:Luminescent Agents; D005486:Flavin Mononucleotide; D049453:Luciferases, Bacterial