Firefly luciferin intermediates

Theoretical Analysis on the Absorption Spectra of Firefly Luciferin Intermediates 61... 

Shibata R, Yoshida Y, Wada N. Filter-photometry of chemiluminescence from firefly luciferin intermediate M420 in deoxygenated dimethyl sulfoxide. J Photosci 2002 9 290-2. 

In the postulated bioluminescence mechanism, firefly luciferin is adenylated in the presence of luciferase, ATP and Mg2+. Luciferyl adenylate in the active site of luciferase is quickly oxygenated at its tertiary carbon (position 4), forming a hydroperoxide intermediate (A). 

Fig. 1.12 Mechanism of the bioluminescence reaction of firefly luciferin catalyzed by firefly luciferase. Luciferin is probably in the dianion form when bound to luciferase. Luciferase-bound luciferin is converted into an adenylate in the presence of ATP and Mg2+, splitting off pyrophosphate (PP). The adenylate is oxygenated in the presence of oxygen (air) forming a peroxide intermediate A, which forms a dioxetanone intermediate B by splitting off AMP. The decomposition of intermediate B produces the excited state of oxyluciferin monoanion (Cl) or dianion (C2). When the energy levels of the excited states fall to the ground states, Cl and C2 emit red light (Amax 615 nm) and yellow-green light (Amax 560 nm), respectively.

One is the concerted decomposition of a dioxetanone structure that is proposed for the chemiluminescence and bioluminescence of both firefly luciferin (Hopkins et al., 1967 McCapra et al., 1968 Shimomura et al., 1977) and Cypridina luciferin (McCapra and Chang, 1967 Shimomura and Johnson, 1971). The other is the linear decomposition mechanism that has been proposed for the bioluminescence reaction of fireflies by DeLuca and Dempsey (1970), but not substantiated. In the case of the Oplopborus bioluminescence, investigation of the reaction pathway by 180-labeling experiments has shown that one O atom of the product CO2 derives from molecular oxygen, indicating that the dioxetanone pathway takes place in this bioluminescence system as well (Shimomura et al., 1978). It appears that the involvement of a dioxetane intermediate is quite widespread in bioluminescence. 

As mentioned earlier (see p. 122) the previously postulated dioxetane intermediate in firefly bioluminescence has been challenged as no 180 is in-corporated in the carbon dioxide released during oxidation of firefly luciferin with 18C>2. In view of the crucial significance of the 180. experiments De Luca and Dempsey 202> rigorously examined the reliability of their tracer method. They conclude from their experiments that all available evidence is in favour of a linear, not a cyclic peroxide intermediate — in contrast to Cypridina bioluminescence where at least part of the reaction proceeds via a cyclic peroxide (dioxetane) as concluded from the incorporation of 180 into the carbon dioxide evolved 202,203). However, the dioxetane intermediate is not absolutely excluded as there is the possibility of a non-chemiluminescent hydrolytic cleavage of the four-membered ring 204>. 

Of the many types of bioluminescence in nature, that of the firefly represents the most thoroughly studied and best understood biological luminescent process. The molecular mechanism of light emission by the firefly was elucidated in the 1960s in which a dioxetanone (a-peroxy lactone) was proposed as an intermediate, formed by the luciferase-catalyzed enzymatic oxidation of the firefly luciferin with molecular oxygen (Scheme 15). This biological reaction constitutes one of the most efficient luminescent processes known to date . Hence, it is not surprising that the luciferin-luciferase system finds wide use... 

Analogously to the firefly luciferin/luciferase system, the general chemiluminescence mechanism postulated for 9-carboxyacridinium derivatives proposes the 1,2-dioxetanone 45 as high-energy intermediate However, this 1,2-dioxetanone is the only intermediate that has not yet been isolated . The cleavage of the peroxidic ring presumably results in the release of CO2 and the formation of an acridan residue in its electronically excited state (Scheme 32). 

Itoh S, Nameda N. Molecular orbital calculations for dioxetane as a part of the intermediate of firefly luciferin. Kagoshima Daigaku Kenkyu Houkoku 1996 38 257-60. 

THEORETICAL ANALYSIS ON THE ABSORPTION SPECTRA OF INTERMEDIATES OF FIREFLY LUCIFERIN IN DEOXYGENATED DIMETHYL SULFOXIDE... 

Oxygen-18 studies of the chemiluminescence of a firefly luciferin analogue have provided evidence which contradicts other earlier reports in that it suggests that the dioxetanone is an important intermediate in light production.304 Spin-statistical contributions in redox chemiluminescence quantum efficiency have been analysed,305 and a striking deuterium isotope effect in phosphorus chemiluminescence has been discovered.800... 

An example of a chemiluminescent molecule in nature is firefly luciferin. The base oxidation of this molecule furnishes a dioxacyclobutanone intermediate that decomposes in a manner analogous to that of 3,3,4,4-tetramethyl-l,2-dioxacyclobutane to give a complex heterocycle, carbon dioxide, and emitted light. 

Chemiluminescence and Bioluminescence.—The function of thiazole and benzothiazole derivatives in the processes of bioluminescence and chemiluminescence continues to form the subject of significant investigations. Extensive contributions have been provided by White et al., ° who have reviewed this interesting and active field. They have shown that the luminescence of firefly luciferins involves intermediates having dioxetan structures. 

Fontes, R., et al. (1998). Dehydroluciferin-AMP is the main intermediate in the luciferin dependent synthesis of Ap4A catalyzed by firefly luciferase. FEBS Lett. 438 190-194. 

The firefly enzyme (EC 1.13.12.7) catalyzes the intermediate formation of D(-)-luciferyl adenylate and pyrophosphate fromD(-)-luciferin and ATP, followed by the oxidative reaction of the acyl adenylate with molecular oxygen to form an enzyme-bound product in the excited... 

In McElroy s lab, we established that the reaction of ATP and luciferin with purified luciferase involves two steps 9 the first forms an active intermediate, later determined to be the adenylate, and the second is the reaction with oxygen, leading to an excited state and light emission. The prompt decline of luminescence over the first minutes was shown to be due to luciferase inhibition, not substrate exhaustion. All evidence indicates that the flash of the firefly is initiated by the introduction of oxygen into the photocytes, triggered by a nerve impulse, which actually does not end on the photocytes, but on adjacent cells.10 12 More recently, nitric oxide (NO)... 

Some years ago I demonstrated that such a biochemical flash can be produced in the test tube.9,15 If oxygen is excluded from a firefly luciferase reaction mixture and then added rapidly back, a bright flash occurs, some 100 to 200 times brighter than the baseline intensity (Fig. 3). This comes from the reaction of the luciferyl adenylate active intermediate accumulated in the absence of oxygen. Note that the decay of the flash is not due to the removal of oxygen, but to the utilization of the luciferase-peroxide intermediate, so the baseline returns to a low level (Fig. 4), defined by the slow rate of reaction of ATP with luciferin. It is well known that the kinetics of firefly flashes are species specific and of functional importance in courtship communication, fixed by the rate constant for the first order decay of the peroxide intermediate formed from the adenylate. 

Oxygen-18 labeling experiments provide mechanistic confirmation for the intervention of a-peroxylactones (2) in luciferin bioluminescence. The first such experiment was performed by DeLuca and Dempsey.77 It was concluded that no a-peroxylactone intermediate was involved in firefly bioluminescence. Instead of the cyclic peroxide path [Eq. (29a)], the tetrahedral intermediate path [Eq. (29b)] was postulated. However, White and co-workers7> observed that in the chemical oxygenation the... 

The luciferase from the North American firefly P. pyralis is a monomeric enzyme (62 kDa) consisting of 550 amino acid residues. The firefly luciferase produces light by the ATP-dependent oxidation of D(-)-luciferin (LH2) (see Scheme 8.3). The reaction involves an enzyme-hound luciferyl adenylate intermediate. The peak light emission occurs at 562 nm (yellow—green, quantum yield of 0.88) in dianionic form between pH 7 and 8 or at 610 nm (red, quantum yield of 0.2) in monoanionic form at pH values below 7. The red shift also occurs in the presence of Zn (2.3 mM) or Cd (12 mM) (58). 

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