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

Abstract

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.

A Conical Intersection Controls the Deactivation of the Bacterial Luciferase Fluorophore

“…Interestingly, the computed emission in THF (that is similar to the measured 2-MeTHF l max ) is not far from the one observed in the luciferase of Vibrio harveyi (490 nm). [15,43] Our confinement hypothesis is supported by a study by Hou et al [23] employing a TD-CAM-B3LYP based hybrid quantum mechanical/molecular mechanical model of such luciferase. It is shown that the fluorescence originates from a p,p* 4a-hydroxy flavin with geometry and charge distribution similar to those of the second Me-FlOH structure in Figure 1 a.…”

“…[8,9] However, when the same cofactors are protein-bound, the confinement [6,7] leads to an enhanced fluorescence emission. [10][11][12][13][14][15] The exact geometry and electronic structure of the emitting conformer and the mechanism preventing the efficient internal conversion occurring in solution are presently unknown.A spectacular manifestation of the effects of protein confinement on flavin cofactors is seen in bacterial luciferases where a 4a,5 flavin adduct is held responsible for the bioluminescence of a vast group of marine eubacteria. In organisms such as Vibrio harveyi, the emission of the fluorophore may be strong enough to cause a blue luminescence of the microbial population that is so intense as to be observable from satellites.…”

“…While these compounds are fluorescent in the protein [15] their fluorescence is weak when unbound. Using femtosecond pump-probe spectroscopy, we found that, in solution, the first singlet excited (S 1 ) state of Et-FlOH decays to the ground (S 0 ) state with several time constants but with a dominant component that has a 500 fs lifetime.…”

“…[7,15,24,30] This suggests that confinement is also responsible for an increased S 1 lifetime in the protein cavity. However, such a mechanism would only be possible if a large change in molecular shape is required for fast internal conversion.…”

Reaction mechanism of surfactant‐sensitized chemiluminescence of bis(2,4,6‐trichlorophyenyl) oxalate and hydrogen peroxide induced by gold nanoparticles

“…Altering the microenvironment of the CL reaction. The cage structure of the micelle is helpful for stabilizing the excited state and preventing it from quenching, thus the CL quantum yield (Φ CL ) is improved (27,28). The CL quantum yield (Φ CL ) is equal to the product of the excitation quantum yield (Φ EX , excited states per molecule reacted) and the emission quantum yield (Φ EM , photons per excited state).…”