Characterization and postulated structure of the primary emitter in the bacterial luciferase reaction

Abstract

An intermediate identifiable as the emitter in bacterial bioluminescence has been demonstrated. The reaction was carried out at 1°C by mixing purified luciferasebound FMN 4a-hydroperoxide with long-chain aldehyde (decanal). Simultaneous kinetic measurements of bioluminescence and absorbance showed that the decay of light emission occurred more rapidly than the appearance of the stable product, oxidized FMN, indicating the formation of a transient intermediate species subsequent to light emission. The same species was found in reaction mixtures examined immediately after light emission was completed. It has a relatively short half-life (7 min at 9°C); the chromophore is postulated to be the luciferase-bound flavin 4a-hydroxide and to decay to the stable product, FMN, by losing water. Both its absorption spectrum (X.,, 360 nm) and its fluorescence emission (X,l, 490 nm) are consistent with the hypothesis that this is the ground state of the primary emitter, the bioluminescent species produced in the reaction.In bioluminescence, a product of the reaction is generated in a singlet excited state, which may either directly emit the characteristic luminescence or, alternatively, transfer its energy to a secondary emitter not necessarily involved in the reaction (1, 2). If the primary excited species is itself the emitter, then the reaction product, now in its ground state after emission, should be identifiable by its fluorescence. However, this species need not be the final reaction product and, therefore, its lifetime may be short, making it elusive.In the bacterial bioluminescent reaction such an emitter has not been previously identified (2, 3). FMN, a product of the reaction and a highly fluorescent molecule, could be the emitter in the reaction, but its fluorescence emission is centered at 530 nm, while the bioluminescence peaks around 490 nm (4, 5). Moreover, luciferase-bound FMN is nonfluorescent (5, 6). Nevertheless, from studies with active flavin analogs having different fluorescence properties, it has been concluded that the emitter is a flavin species (3, 4, 7).Additional information became available with the isolation and identification of the luciferase-bound flavin 4a-hydroperoxide (8-10). As an intermediate in the oxidation of FMNH2 by oxygen (in the absence of aldehyde), this species has a half-life of about 1 hr at 2°C (11, 12) and, under appropriate conditions, exhibits a high quantum yield fluorescence with an emission closely matching that of the bioluminescence (13). However, this peroxyflavin cannot be the emitter either, because it has not yet reacted with aldehyde in the catalytic pathway. The luciferase-bound flavin 4a-hydroxide should have a similar fluorescence and could be produced in this reaction in the singlet excited state (14, 15). The experiments described below provide evidence for the occurrence and emitter role of the hydroxyflavin chromophore. (11,21,22). FMN (3 x 10-4 M, final concentration) was added to 6 mg of luciferase in 0.7 ml of 0.35 M phosphate buffer (pH 7) and ...

Spectral detection of an intermediate preceding the excited state in the bacterial luciferase reaction

“…The expected product, then, is the ground state of this species, the luciferase-bound flavin 4a-hydroxide (IV), previously identified by Kurfurst et al (1984Kurfurst et al ( ,1987. A fluorescent transient described by Matheson and Lee (1983) is most likely the same species.…”

Yellow light emission of Vibrio fischeri strain Y-1: purification and characterization of the energy-accepting yellow fluorescent protein.

“…Two lines of evidence have suggested that the light emitted comes from the singlet excited state of an enzyme-bound flavin intermediate formed in the luciferase reaction for Vibrio harveyi, both in vivo and in vitro: (i) Cline and Hastings found that several mutants of V. harveyi having altered luciferase reaction kinetics also had altered bioluminescence emission spectra that had been red-shifted as much as 12-15 nm both in vivo and in vitro (2,3), and (it) Mitchell and Hastings showed that the spectrum of light emitted in vitro is strongly shifted by alterations in the isoalloxazine nucleus ofthe flavin (4). It has been suggested that the emitter in the reaction catalyzed by the V. harveyi enzyme in vitro is a carbon-4a-substituted flavin intermediate, possibly the 4a-hydroxyflavin (5)(6)(7).…”

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.

“…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).…”