The chemiluminescence of a Cypridina luciferin analogue

McCapra, Frank; Chang, Yew C.

doi:10.1039/c19670001011

Cited by 48 publications

(56 citation statements)

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“…In many studies of these bioluminescence systems, it is stated that the excited coelenteramide amide anion is the primary excited state product. This belief arises from earlier reports such as by McCapra and Chang (35), who investigated the chemiluminescence of an imidazopyrazinone derivative in an aprotic solvent with strong base and provided convincing evidence for a coelenteramide anion as the primary excited state. However, in photoprotein bioluminescence, if the coelenteramide anion were the primary excited state product, it would be energetically infeasible for it to populate the neutral excited state at higher energy.…”

Section: Methodsmentioning

confidence: 98%

Crystal structure of obelin after Ca²⁺-triggered bioluminescence suggests neutral coelenteramide as the primary excited state

Liu

Stepanyuk

Vysotski

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

104

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The crystal structure at 1.93-Å resolution is determined for the Ca 2؉ -discharged obelin containing three bound calcium ions as well as the product of the bioluminescence reaction, coelenteramide. This finding extends the series of available spatial structures of the ligand-dependent conformations of the protein to four, the obelin itself, and those after the bioluminescence reaction with or without bound Ca 2؉ and͞or coelenteramide. Among these structures, global conformational changes are small, typical of the class of ''calcium signal modulators'' within the EF-hand protein superfamily. Nevertheless, in the active site there are significant repositions of two residues. The His-175 imidazole ring flips becoming almost perpendicular to the original orientation corroborating the crucial importance of this residue for triggering bioluminescence. Tyr-138 hydrogen bonded to the coelenterazine N1-atom in unreacted obelin is moved away from the binding cavity after reaction. However, this Tyr is displaced by a water molecule from within the cavity, which now forms a hydrogen bond to the same atom, the amide N of coelenteramide. From this observation, a reaction scheme is proposed that would result in the neutral coelenteramide as the primary excited state product in photoprotein bioluminescence. From such a higher energy state it is now energetically feasible to account for the shorter wavelength bioluminescence spectra obtained from some photoprotein mutants or to populate the lower energy state of the phenolate anion to yield the blue bioluminescence ordinarily observed from native photoproteins.coelenterazine ͉ photoprotein ͉ EF hand ͉ luciferase ͉ aequorin

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Section: Methodsmentioning

confidence: 98%

Crystal structure of obelin after Ca²⁺-triggered bioluminescence suggests neutral coelenteramide as the primary excited state

Liu

Stepanyuk

Vysotski

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

104

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“…The contributions of 180 in the contaminant CO2 to the total 180 found in experiments 2, 4, and 6 are calculated as 65%, 71%, and 73%, respectively, by Eqs. 7 and 8. These figures indicate that the data of experiments 2, 4, and 6 given in Table 1, for experiments in H2180 medium with 1602 gas, are hardly meaningful in the interpretation of the reaction mechanism.…”

Section: Resultsmentioning

confidence: 99%

Source of oxygen in the CO ₂ produced in the bioluminescent oxidation of firefly luciferin

Shimomura

Goto

Johnson

1977

Proc. Natl. Acad. Sci. U.S.A.

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Incorporation of 180 into the CO2 produced in the bioluminescent oxidation of firefly luciferin was studied. In H2160 medium with 1802 gas, the product CO2 contained up to 75% C'60180, showing that one 0 of the product CO2 arose from the 02 that oxidized luciferin. This result is consistent with a dioxetane mechanism. Analysis of the mass spectral data of the CO2 obtained in high-enrichment H2180 medium with 1602 gas indicated the presence of about 20% Bioluminescence and chemiluminescence of the luciferins of the firefly, the ostracod Cypridina, and the sea pansy Renilla are similar in that all require molecular oxygen and produce CO2 as a product (1-3). A mechanism that involves a dioxetane intermediate, scheme 1, has been proposed for the luminescence of both firefly luciferin (4-6) and Cypridina luciferin (7). / Here, one 0 of the product CO2 originates from molecular oxygen. To test this scheme, DeLuca and Dempsey studied the labeling of the product CO2 with 180 in the bioluminescence reaction of firefly luciferin (8, 9). Their data indicated that one o of the CO2 was labeled when the reaction was carried out in H2180 medium with 1602 gas, but not in H2160 medium with 1802 gas. They proposed reaction scheme 2 instead of 1.In chemiluminescence of firefly luciferin, the data reported by White et al. (10) were consistent with scheme 1 but did not completely rule out scheme 2 (11), in contrast to the data of DeLuca et al. (12), which supported scheme 2 but failed to rule out scheme 1. Studies on the bioluminescence of Cypridina Abbreviation: m/e, mass-to-charge ratio. 2799 luciferin by labeling product CO2 with 180 fully supported scheme 1 (13-15). In regard to Renilla luciferin, however, one 0 of the product CO2 was reported to arise from solvent H120 in both bioluminescence (3) and chemiluminescence (12), which would seem odd in view of the structural similarity between Renilla luciferin and Cypridina luciferin. The present study unambiguously supports scheme 1, but not scheme 2, for the bioluminescence of firefly luciferin. Moreover, present data cast a serious doubt on the validity of previously reported data (3) Firefly Luciferase. Purification was by the following procedure, hitherto unpublished. Acetone powder prepared from 10 g of lanterns of freeze-dried Photinus fireflies (collected at Princeton, NJ) was mixed with 200 ml of 25 mM Tris-HC1 buffer containing 1 mM EDTA (pH 7.9 at 50) and the pH of the mixture was readjusted to 7.9 with Tris, then centrifuged. The precipitate was mixed with 50 ml of the same buffer and the pH was adjusted to 7.9; then the mixture was centrifuged again. The supernatants were combined and fractionated with (NH4)2SO4; the fraction precipitated between 0.32 and 0.58 saturation was saved. This preparation was purified on a column of Sephadex G-150 (Pharmacia) (2.6 X 80 cm) equilibrated with 10 mM Tris-HCI buffer containing 2 mM EDTA and 10% (NH4)2SO4 (pH 7.75), and finally recrystallized three times by dialysis as described by Green and McElroy (17).The luciferase...

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“…Such a compound would be very unstable in free solution, but in the protein it appears to be frozen in place via a H-bond network to amino acid residues comprising the binding cavity. Model chemiluminescence studies with coelenterazine analogues had shown such a peroxide to be an intermediate, closing to a dioxetanone in the reaction pathway (8). The free energy produced by decarboxylation of this dioxetanone around 70 kcal/mol is sufficient to account for the energy of the photons of blue bioluminescence.…”

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confidence: 99%

Picosecond Fluorescence Relaxation Spectroscopy of the Calcium-Discharged Photoproteins Aequorin and Obelin

et al. 2009

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Addition of calcium ions to the Ca 2þ -regulated photoproteins, such as aequorin and obelin, produces a blue bioluminescence originating from a fluorescence transition of the protein-bound product, coelenteramide. The kinetics of several transient fluorescent species of the bound coelenteramide is resolved after picosecond-laser excitation and streak camera detection. The initially formed spectral distributions at picosecond-times are broad, evidently comprised of two contributions, one at higher energy (∼25 000 cm -1 ) assigned as from the Ca 2þ -discharged photoprotein-bound coelenteramide in its neutral state. This component decays much more rapidly (t 1/2 ∼ 2 ps) in the case of the Ca 2þ -discharged obelin than aequorin (t 1/2 ∼ 30 ps). The second component at lower energy shows several intermediates in the 150-500 ps times, with a final species having spectral maxima 19 400 cm -1 , bound to Ca 2þ -discharged obelin, and 21 300 cm -1 , bound to Ca 2þ -discharged aequorin, and both have a fluorescence decay lifetime of 4 ns. It is proposed that the rapid kinetics of these fluorescence transients on the picosecond time scale, correspond to times for relaxation of the protein structural environment of the binding cavity.Bioluminescent animals are found in a variety of types occurring both terrestrially and in the ocean. More often than not, the chemistry of their light emission processes and the proteins involved are found quite unrelated. Well-studied cases, for example, are the bioluminescence of the firefly, which involves ATP and a substrate firefly luciferin, a benzthiozole derivative, and that of the photoprotein aequorin from the bioluminescent jellyfish Aequorea, which is triggered for light emission by Ca 2þ . Coelenterazine, an imidazopyrazinone derivative (Figure 1), is the luciferin (a generic term for the substrate) involved in many marine bioluminescent systems, including the ones subject to this present study, the Ca 2þ -regulated photoproteins, aequorin and obelin from the hydrazoan Obelia (1-3).A significant advance in uncovering the mechanism of bioluminescence from aequorin and obelin resulted from the determination of the high-resolution spatial structure of the two photoproteins (4-6). The coelenterazine was revealed residing in an internal cavity substituted with a peroxy group ( Figure 1B), as long suspected from earlier indirect evidence (7). Such a compound would be very unstable in free solution, but in the protein it appears to be frozen in place via a H-bond network to amino acid residues comprising the binding cavity. Model chemiluminescence studies with coelenterazine analogues had shown such a peroxide to be an intermediate, closing to a dioxetanone in the reaction pathway (8). The free energy produced by decarboxylation of this dioxetanone around 70 kcal/mol is sufficient to account for the energy of the photons of blue bioluminescence.Aequorin and obelin are EF-hand proteins belonging to the large family of Ca 2þ -binding proteins. They each contain three Ca 2þ -ligati...

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The chemiluminescence of a Cypridina luciferin analogue

Cited by 48 publications

References 0 publications

Crystal structure of obelin after Ca²⁺-triggered bioluminescence suggests neutral coelenteramide as the primary excited state

Crystal structure of obelin after Ca²⁺-triggered bioluminescence suggests neutral coelenteramide as the primary excited state

Source of oxygen in the CO ₂ produced in the bioluminescent oxidation of firefly luciferin

Picosecond Fluorescence Relaxation Spectroscopy of the Calcium-Discharged Photoproteins Aequorin and Obelin

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The chemiluminescence of a Cypridina luciferin analogue

Cited by 48 publications

References 0 publications

Crystal structure of obelin after Ca2+-triggered bioluminescence suggests neutral coelenteramide as the primary excited state

Crystal structure of obelin after Ca2+-triggered bioluminescence suggests neutral coelenteramide as the primary excited state

Source of oxygen in the CO 2 produced in the bioluminescent oxidation of firefly luciferin

Picosecond Fluorescence Relaxation Spectroscopy of the Calcium-Discharged Photoproteins Aequorin and Obelin

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Crystal structure of obelin after Ca²⁺-triggered bioluminescence suggests neutral coelenteramide as the primary excited state

Crystal structure of obelin after Ca²⁺-triggered bioluminescence suggests neutral coelenteramide as the primary excited state

Source of oxygen in the CO ₂ produced in the bioluminescent oxidation of firefly luciferin