Bacterial bioluminescence. Quantum yields and stoichiometry of the reactants reduced flavin mononucleotide, dodecanal, and oxygen, and of a product hydrogen peroxide

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A blue fluorescence protein has been isolated and purified from extracts of the luminous bacterium Photobacterium phosphoreum. It is a single polypeptide of molecular weight 22,000 with absorption maxima at 274 and 418 nm. It is efficiently fluorescent (OF 0.45), with a fully corrected spectral maximum (476 nm) and distribution identical to the in vivo bioluminescence from this same type of bacterium. At low concentration this fluorescence shifts towards the red and becomes identical to the in vitro bioluminescence emission. This spectral shift apparently results from a change in the protein pulled by dissociation of the chromophore . If the blue fluorescence protein is included in the in vitro bioluminescence reaction with reduced FMN, oxygen, aldehyde, and luciferase (P. phosphoreum), the bioluminescence spectrum is shifted towards the blue from its maximum at 490 nm to one at 476 nm, where it is again identical in all respects to the in vivo bioluminescence spectrum. This is accompanied by an increase in the initial light intensity by an order of magitude at saturating levels of blue fluorescence protein, and the specific light yield of the luciferase is increased 4-fold. It is suggested that the blue fluorescence protein acts as a sensitizer of the bacterial bioluminescence reaction. (14) claimed that the emitter must be some sort of flavin-derived species (2), three proposals for its structure were put forward, supported almost solely by the similarity of the fluorescence of model compounds to the bioluminescence spectra. Eley et al. (12) (17) proposed an FMNH2 molecule substituted in the 4a-position.It had been tacitly assumed that the mechanism of reaction and identity of the emitter are the same in vivo as in vitro. The possibility of a difference has been raised by the recent discovery of a bacterial type emitting at 545 nm (18). In this paper we show that a protein-bound chromophore can be isolated from extracts of the bioluminescent bacteria P. phosphoreum that is closely associated with luciferase and that fulfills all the conditions to qualify it as the in vivo emitter. MATERIALS AND METHODSThe bacterium Beneckea harveyi, strain 392 in the classification scheme of Reichelt and Baumann (19), previously designated "MAV,"' was obtained from J. W. Hastings (Harvard University). The type "A-13" was isolated from the light organ of the "silver macrourid" fish by J. Paxton (Australian Museum) and has been identified as Photobacterium phosphoreum (J. Fitzgerald, private communication). The type Photobacterium fischeri, strain 399, was obtained from F. H. Johnson (Princeton University). The bacteria were grown and the luciferase and FMN were purified as described (20,21). The blue fluorescence protein was routinely assayed by its fluorescence intensity at 470 nm when excited at 420 nm, with an Aminco-Bowman Spectrofluorimeter. Luciferase activity was determined with a digital photometer, designed and constructed by G. J. Faini, which was calibrated for absolute photon sensitivity with the luminol ch...

Section: Methodsmentioning

confidence: 99%

Isolation of the in vivo emitter in bacterial bioluminescence

Gast

Lee

1978

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“…A reaction mechanism involving the oxidation of a single FMNH2 and the concomitant oxidation of aldehyde was thus proposed (6,10) and has been supported by a variety of experimental results since then (1,11), including the demonstration that aldehyde is indeed oxidized to acid (12)(13)(14)(15). However, based on the report that the quantum yield with respect to FMNH2 is approximately half that for aldehyde, it again was proposed that two reduced flavins are required per catalytic cycle (16,17 MATERIALS AND METHODS Bacterial luciferase was purified as previously described (18) from Photobacterium fischeri, Beneckea harveyt (19), and from dark mutants of B. harveyi, designated as M16 and M1S, which require exogenous aldehyde for bioluminescence. Enzyme concentrations were determined spectrophotometrically using a weight absorptivity of 0.94 (0.1%, 1 cm) at 280 nm, and a molecular weight of 79,000 (20).…”

mentioning

confidence: 94%

“…A reaction mechanism involving the oxidation of a single FMNH2 and the concomitant oxidation of aldehyde was thus proposed (6, 10) and has been supported by a variety of experimental results since then (1, 11), including the demonstration that aldehyde is indeed oxidized to acid (12-15). However, based on the report that the quantum yield with respect to FMNH2 is approximately half that for aldehyde, it again was proposed that two reduced flavins are required per catalytic cycle (16,17 based on an absorptivity of 12,500 M-1 cm-1 at 445 nm (23). 2,2-Bis(hydroxymethyl)-2,2',2"-nitrilotriethanol (BisTris) and dithiothreitol were obtained from Sigma, decanal from Aldrich, bovine serum albumin from Pentex, dithionite (sodium hydrosulfite) from Mallinckrodt.…”

Section: Introductionmentioning

confidence: 99%

Bacterial luciferase requires one reduced flavin for light emission.

Becvar¹,

Hastings²

1975

Recent reports revive a hypothesis that the bacterial bioluminescence reaction involves two reduced flavin mononucleotide molecules per enzyme turnover. A twoflavin mechanism requires that the two flavins bind simultaneously or sequentially to the same or different sites on luciferase during a catalytic cycle. Measurements using equilibrium techniques show that the luciferase dimer has only a single reduced flavin binding site. (4) indicated that the bioluminescent reaction had a second order dependence on FMNH2 concentration, again suggesting the involvement of two flavins.Some years later, when the cad heterodimeric structure of luciferase was elucidated (5), the possibility that two reduced flavins might be required for activity could be envisioned in more specific terms. However, binding site determinations by a kinetic method showed that luciferase possesses only a single FMNH2 binding site per dimer (6). Furthermore, chemical modification studies (7,8) and mutant analyses (9) indicated that only the a subunit participates in the catalytic steps. A reaction mechanism involving the oxidation of a single FMNH2 and the concomitant oxidation of aldehyde was thus proposed (6, 10) and has been supported by a variety of experimental results since then (1, 11), including the demonstration that aldehyde is indeed oxidized to acid (12-15). However, based on the report that the quantum yield with respect to FMNH2 is approximately half that for aldehyde, it again was proposed that two reduced flavins are required per catalytic cycle (16,17 based on an absorptivity of 12,500 M-1 cm-1 at 445 nm (23). 2,2-Bis(hydroxymethyl)-2,2',2"-nitrilotriethanol (BisTris) and dithiothreitol were obtained from Sigma, decanal from Aldrich, bovine serum albumin from Pentex, dithionite (sodium hydrosulfite) from Mallinckrodt. Absorbance values were read with a Cary 15 spectrophotometer, circular dichroism spectra with a Jasco J-20 spectropolarimeter. Fig. 1 gives circular dichroism (CD) spectra for free FMNH2, free luciferase, and an equimolar mixture of FMNH2 and luciferase, all three in the presence of dithionite. The relatively large signal at 370 nm for the mixture is indicative of a complex between reduced flavin and luciferase. The stoichiometry of this complex was examined by the method of continuous variation of mole fraction according to job (24). When equimolar solutions of FMNH2 and luciferase are mixed in different proportions, the CD signal at 370 nm varies as a function of the relative mole fraction of the reactants (Fig. 2). Maximum signal develops for a mole fraction of flavin (and enzyme) near 0.5, indicating that luciferase has one binding site for FMNH2. Although these data are not ideal for the calculation of a dissociation constant Kd for the E:FMNH2 complex, they are consistent with the published value of 0.8 ,M (6). RESULTS AND DISCUSSION

“…First, it must be there in the reaction, either added to it or identified as a product (at the very least identified as a reasonable hypothetical product). Second, it must have a high fluorescence efficiency sufficient to account for the quantum yield ofthe bioluminescence reaction [for example, at least 0.1 in the case of the reaction of FMNH2 with the luciferase from Photobacteriumfischeri (8)]. Finally, the fluorescence spectral distribution must be the same as that ofthe bioluminescence.…”

mentioning

confidence: 99%

Bacterial bioluminescence: Spectral study of the emitters in the in vitro reaction

Matheson

Lee

Müller

1981

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Transient fluorescent species are.observed in the bioluminescent reactions of three reduced flavin mononucleotides with aliphatic aldehydes and. oxygen, catalyzed by bacterial luciferase. In each case the. fluorescence spectral distribution. is sims flar to that of the bioluminescence but is readily distinguishable from it on the basis of a significantly greater signal strength. The corrected bioluminescence. maxima using Beneckea Iharveyi luciferase are 479 nm (iso-FMNH ), 490 nm (FMNHi), and 560 nm