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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 ...

Here we report the cDNA-deduced amino-acid sequence of l-amino-acid oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FADdependent ophidian LAAOs to FAD-dependent oxidases such as monoamine oxidases, d-amino-acid oxidases and tryptophan 2-monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide-binding fold with monoamine oxidases, tryptophan 2-monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze-and pH-induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl 2 , prevent pH-induced inactivation. LAAO exhibits typical flavoprotein oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.