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...

Abstract— A new strain of the yellow‐light emitting bacterium Vibrio fischeri Y‐lb, possessing a higher fraction of its emission in the yellow, was isolated and characterized. The in vivo yellow to blue ratio (Y/B) increases during growth in parallel with the autoinduction of luciferase, reaching a value above 5 at 17°C. This Y/B increase is attributed to concomitant increases in the intracellular levels of both luciferase and the specific protein associated with yellow emission, the yellow fluorescent protein (YFP), and to the association of the two proteins at higher concentrations. The yellow emission is rapidly but reversibly lost by brief exposures to a temperature of 27°C (5 s), and irreversibly so at 40°C, attributed to dissociation of the two proteins. A concentrated crude extract emits almost exclusively yellow light in the in vitro reaction, and theY/B ratio decreases with dilution. The Y/B ratio in vitro is also dependent on the aldehyde chain length; experiments with aldehyde analogs suggest that the aldehyde binding site of luciferase must be occupied for YFP to stimulate light emission.