A TIME-DEPENDENT BACTERIAL BIOLUMINESCENCE EMISSION-SPECTRUM IN AN INVITRO SINGLE TURNOVER SYSTEM - ENERGY-TRANSFER ALONE CANNOT ACCOUNT FOR THE YELLOW EMISSION OF VIBRIO-FISCHERI Y-1

Yellow fluorescent protein (YFP), which has a bound FMN, was isolated from the marine bacterium Vibrio fischeri strain Y-1b. 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 [YFP] (from 0.01 sec-1 with no YFP to a maximum of ≈0.1 sec-1 at 4°C) but exhibiting a saturation behavior. The data can be interpreted by assuming the interaction of YFP with the peroxyhemiacetal intermediate in the luciferase reaction to form an unstable new complex whose breakdown gives the yellow emitter in its excited state. This simple model fits well the data at [YFP] < 15 μM. The results indicate that a single primary excited state cannot be responsible for the blue and the yellow emissions. (.