Luciferin oxygen

Firefly luciferase (E.C. 1.13.12.7 - Photinus luciferin oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolyzing). 

Chemiluminescence is also obtained by anionic autooxidation of (41) with oxygen ia alkaline dimethyl sulfoxide (DMSO) (216). Qc has been reported to be 10% and ketone (43) and CO2 are obtained. Several analogues of luciferin have been prepared that are also chemiluminescent when they react with oxygen ia alkaline DMSO (62). 

In the first step, luciferin is converted into luciferyl adenylate by ATP in the presence of Mg2+. In the second step, luciferyl adenylate is oxidized by molecular oxygen resulting in the emission of yellow-green light, of which the mechanism is discussed in Sections 1.1.6 and 1.1.7. Both steps, (1) and (2), are catalyzed by luciferase. The reaction of the first step is slower than that of the second step, thus the first step is the rate-limiting step. 

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.

In the luminescence reaction of firefly luciferin (Fig. 1.12), one oxygen atom of the product CO2 is derived from the molecular oxygen while the other originates from the carboxyl group of luciferin. In the chemiluminescence reaction of an analogue of firefly luciferin in DMSO in the presence of a base, the analysis of the product CO2 has supported the dioxetanone pathway (White et al., 1975). 

Under illumination, some of the luciferin molecules (LnH) that absorbed photons are changed into free radicals (Ln ), probably at carbon-2 of the imidazopyrazinone ring. The free radical instantly binds with an oxygen molecule to form a peroxide radical (LnOO ), an extremely fast reaction (k = 109M 1 s"1 Pryor, 1976). The peroxide radical formed reacts with a luciferin molecule, generating a new free radical of luciferin and a luciferin peroxide anion (LnOO"),... 

The fluorescent compound F, a luciferin, emits blue light (Amax 476 nm Fig. 3.2.4) in the presence of molecular oxygen and the protein P, a luciferase. In the luminescence reaction, F is changed into an oxidized form (structure 8, Fig. 3.2.6). The luminescence reaction is highly sensitive to pH, with a narrow optimal range around pH 7.8 (Fig. 3.2.2) the optimum salt concentration is 0.15 M for NaCl... 

The bioluminescence reaction of Oplophorus is a typical luciferin-luciferase reaction that requires only three components luciferin (coelenterazine), luciferase and molecular oxygen. The luminescence spectrum shows a peak at about 454nm (Fig. 3.3.1). The luminescence is significantly affected by pH, salt concentration, and temperature. A certain level of ionic strength (salt) is necessary for the activity of the luciferase. In the case of NaCl, at least 0.05-0.1 M of the salt is needed for a moderate rate of light emission, and about 0.5 M for the maximum light intensity. 

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. 

Harvey (1917) noted that the fresh arm photophores of Watasenia scintillans do not show a luciferin-luciferase reaction, and Shoji (1919) using a gas chamber of purified hydrogen demonstrated that molecular oxygen is needed for the luminescence. 

Fig. 6.3.5 A reaction scheme proposed by Tsuji (2002) for the Watasenia bioluminescence. The proposed mechanism involves the adenylation of luciferase-bound luciferin by ATP, like in the bioluminescence of fireflies. However, the AMP group is split off from luciferin before the oxygenation of luciferin, differing from the mechanism of the firefly bioluminescence. Thus the role of ATP in the Watasenia bioluminescence reaction remains unclear. Reproduced with permission from Elsevier.

Harvey (1952) demonstrated the luciferin-luciferase reaction with O. phosphorea collected at Nanaimo, British Columbia, Canada, and with O. enopla from Bermuda. McElroy (1960) partially purified the luciferin, and found that the luminescence spectrum of the luciferin-luciferase reaction of O. enopla is identical to the fluorescence spectrum of the luciferin (A.max 510 nm), and also that the luciferin is auto-oxidized by molecular oxygen without light emission. Further investigation on the bioluminescence of Odontosyllis has been made by Shimomura etal. (1963d, 1964) and Trainor (1979). Although the phenomenon is well known, the chemical structure of the luciferin and the mechanism of the luminescence reaction have not been elucidated. 

In addition to the reactions of luciferin shown in Fig. 7.2.3, Trainor (1979) made an interesting discovery that arylsulfatase from Pattela vulgata (Sigma) converts luciferin into a violet compound (treatment conditions pH 5.05, 37°C, 4 hr). The compound showed absorption peaks at 344 nm and 560 nm (Fig. 7.2.7), and the peaks shifted reversibly to 347 nm and 547 nm in acid, and to 364 nm and 720 nm in an alkaline solution. The violet compound was also obtained from the pink compound by treatment with arylsulfatase, or from luciferin by heating in 50% trifluoroacetic acid at 100°C for 1 hr, followed by an addition of oxygenated water. These results, together with the... 

Luciferase-catalyzed luminescence of luciferin. Odontosyllis luciferin emits light in the presence of Mg2+, molecular oxygen and luciferase. The relationship between the luminescence intensity and the pH of the medium shows a broad optimum (Fig. 7.2.8). The luminescence reaction requires a divalent alkaline earth ion, of which Mg2+ is most effective (optimum concentration 30 mM). Monovalent cations such as Na+, K+, and NH have little effect, and many heavy metal ions, such as Hg2+, Cu2+, Co2+ and Zn2+, are generally inhibitory. The activity of crude preparations of luciferase progressively decreases by repeated dialysis and also by concentrating the solutions under reduced pressure. However, the decreased luciferase activity can be completely restored to the original activity by the addition of 1 mM HCN (added as KCN). The relationship between the concentration of HCN and the luciferase activity is shown in Fig. 7.2.9. Low concentrations of h and K3Fe(CN)6 also enhance luminescence, but their effects are only transient. 

Luminescence reaction (Bellisario et al., 1972). Mixing the luciferin, luciferase and H2O2 results in an emission of light, regardless of the presence or absence of molecular oxygen. The in vitro luminescence with partially purified luciferin and luciferase (A.max 503 nm) was spectrally similar to the in vivo luminescence from freshly exuded slime (A.max 507 nm) (Fig. 7.3.2). However, Ohtsuka et al. (1976) reported that the emission maximum was found at 490 nm when a pure sample of luciferin was used. 

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