A time-dependent bacterial bioluminescence emission spectrum in an in vitro single turnover system: energy transfer alone cannot account for the yellow emission of Vibrio fischeri Y-1.

Abstract

Yellow fluorescent protein (YFP), which has a bound FMN, was isolated from the marine bacterium Vibrio ficheri strain Y-lb. Its presence in a luciferase [alkanal monooxygenase (FMN-linked); alkanal, reduced-FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] reaction mixture causes a striking color change, and an increase in bioluminescence intensity, as well as a faster rate of intensity decay, so that the quantum yield is not changed. The emission spectrum shows two distinct color bands, one at 490 nm attributed to the unaltered emission of the luciferase system, the other peaking in the yellow around 540 nm due to YFP emission. The kinetics of the two color bands differ, so the spectrum changes with time. The yellow emission reaches its initial maximum intensity later than the blue, and then both blue and yellow emissions decay exponentially with nearly the same pseudo-first-order rate constants, linearly dependent on In contrast to most species of bioluminescent bacteria, which emit blue light, a strain (Y-1) of Vibrio fischeri emitting yellow light was isolated some years ago (1); a shoulder at 490 nm in the emission spectrum (see Fig. 1A) corresponds to the peak of bioluminescence of wild-type V. fischeri. The remarkable large spectral shift involved (from Amax = 490 to Amax = 540 nm) was found to be due to the presence, in these bacteria, of a yellow fluorescent protein (YFP) (2), in which the emitting chromophore was identified as FMN (3)(4)(5).Bacterial bioluminescence accompanies the oxidation of FMNH2 and a long-chain aldehyde by molecular oxygen, catalyzed by the enzyme luciferase [alkanal monooxygenase (FMN-linked); alkanal, reduced-FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] (6). Luciferase extracted from the Y-1 strain does not appear to differ significantly from normal blue-emitting V. fischeri luciferase. It has similar molecular weight and subunit structure and a similarly slow catalytic cycle; a single enzymatic turnover takes =100 sec at 40C. In addition, the light emitted in the reaction of FMNH2 catalyzed by Y-1 luciferase peaks at the characteristic 490 nm (see Fig. 1B; ref. 2). However, the presence of YFP in the reaction mixture-but not its chromophore FMN or the apoprotein alone-brings about the yellow emission (see Fig. 1B).** The relative magnitude of the bands at 490 and 540 nm was found to depend strongly on the concentration of YFP and temperature (2), suggesting a reversible association between luciferase and YFP.It has been proposed that the color shift can be attributed to energy transfer (1,4,5). For example, the excited chromophore of the luciferase reaction, which is responsible for the blue emission and has been identified as 4a-hydroxyflavin (in Vibrio harveyi; ref. 7), would donate its energy to YFP, thereby generating excited YFP, which would then emit its yellow fluorescence. Alternatively, it has been suggested that the excited states of the two chromophores could be populated independently (6).The kinetic and spec...

The mechanism of electronic excitation in the bacterial bioluminescent reaction

“…For more detailed determination of the mechanism of formation of this spectrum, Eckstein et al 72 studied the kinetics of reaction (1) in the presence of YFP. They found a complex and, what is more important, different patterns of variation of the time decay rates for the`blue' and`yellow-green' bioluminescence components.…”

Protein-protein complexation in bioluminescence

“…), and K D = 0.7 µmol/L, evidence that YFP (and LumP in a less strong manner) has a catalytic effect on the luminescence reaction (Eckstein et al, 1990;Petushkov et al, 1995Petushkov et al, , 1996aPetushkov et al, , 1996b.…”

Properties of 4a-hydroxy-4a,5-dihydroflavin radicals in relation to bacterial bioluminescence

“…35 In addition, kinetic datags with FMN suggest that the presence of the latter may alter the chemiexcitation mechanism altogether. It has been known for some time that in the presence of lumazine proteins32 the bioluminescence is blue-shifted while binding to FMN-containing fluorescent protein results in yellow l~minescence.~~ In the former case an energy transfer from the emitting flavin pseudobase excited state would have to be an uphill process, thus not very l i k e l~?~ In the latter case, spectral studies on chemically modified dihydroflavin substrates also seem to argue against a FBrster type energy transfer.…”