Bioluminescence reactions

Firefly. Firefly luciferase (EC 1.13.12.7) is a homodimeric enzyme (62 kDa subunit) that has binding sites for firefly luciferin and Mg ATP . Amino acid sequence analysis has iadicated that beetle luciferases evolved from coen2yme A synthetase (206). Firefly bioluminescence is the most efficient bioluminescent reaction known, with Qc reported to be 88% (5), and at 562 nm (56). At low pH and ia the presence of certain metal ions (eg, Pb ", ... 

Genes lux) encoding luciferase and the other proteias iavolved ia the bioluminescent reaction have been cloned. PuxA. and luxP genes code for the luciferase subunits, and the fatty acid reductase complex is coded by the luxCDE genes. 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

The sharp flash in the firefly bioluminescence reaction (Fig. 1.6) is due to the formation of a strongly inhibitory byproduct in the reaction. The inhibitor formed is dehydroluciferyl adenylate, having the structure shown below at left. In the presence of coenzyme A (CoA), however, this inhibitory adenylate is converted into dehydroluciferyl-CoA, a compound only weakly inhibitory to luminescence. Thus, an addition of CoA in the reaction medium results in a long-lasting, high level of luminescence (Airth et al., 1958 McElroy and Seliger, 1966 Ford et al., 1995 Fontes et al., 1997, 1998). 

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.

This finding by Branchini et al. (2002) clearly indicates that 5,5-dimethyloxyluciferin is able to emit the two different colors. This conclusion, however, does not rule out the involvement of the enolized oxyluciferin in the bioluminescence reaction of firefly. 

Orlova et al. (2003) theoretically studied the mechanism of the firefly bioluminescence reaction on the basis of the hybrid density functional theory. According to their conclusion, changes in the color of light emission by rotating the two rings on the 2-2 axis is unlikely, whereas the participation of the enol-forms of oxyluciferin in bioluminescence is plausible but not essential to explain the multicolor emission. They predicted that the color of the bioluminescence depends on the polarization of the oxyluciferin molecule (at its OH and O-termini) in the microenvironment of the luciferase active site the... 

Contrary to the dioxetanone pathway, DeLuca and Dempsey (1970) proposed a mechanism of the bioluminescence reaction that involves a multiple linear bond cleavage of luciferin peroxide... 

However, the linear bond cleavage hypothesis of the firefly bioluminescence was made invalid in 1977. It was clearly shown by Shimomura et al. (1977) that one O atom of the CO2 produced is derived from molecular oxygen, not from the solvent water, using the same 180-labeling technique as used by DeLuca and Dempsey. The result was verified by Wannlund et al. (1978). Thus it was confirmed that the firefly bioluminescence reaction involves the dioxetanone pathway. Incidentally, there is currently no known bioluminescence system that involves a splitting of CO2 by the linear bond cleavage mechanism. 

Fig. 1.14 Relationship between the incorporation of 180 into product CO2 and the amount of luciferin used, in the bioluminescence reaction of Cypridina luciferin catalyzed by Cypridina luciferase. The reactions were carried out in H2160 medium with 1802 gas (solid line) in H2lsO medium with 16C>2 gas (dashed line) and the control experiment of the latter using C16C>2 gas instead of luciferin and luciferase (dotted line), all in 20 mM glycylglycine buffer, pH 7.8, containing 40 mM NaCl. From Shimomura and Johnson, 1973a. Reproduced with permission from Elsevier.

Fig. 2.1 Mechanism of the bacterial bioluminescence reaction. The molecule of FMNH2 is deprotonated at N1 when bound to a luciferase molecule, which is then readily peroxidized at C4a to form Intermediate A. Intermediate A reacts with a fatty aldehyde (such as dodecanal and tetradecanal) to form Intermediate B. Intermediate B decomposes and yields the excited state of 4a-hydroxyflavin (Intermediate C) and a fatty acid. Light (Amax 490 nm) is emitted when the excited state of C falls to the ground state. The ground state C decomposes into FMN plus H2O. All the intermediates (A, B, and C) are luciferase-bound forms. The FMN formed can be reduced to FMNH2 in the presence of FMN reductase and NADH.

The reported quantum yields of the long-chain aldehydes in the luminescence reaction catalyzed by P. fischeri luciferase are 0.1 for dodecanal with the standard I (Lee, 1972) 0.13 for decanal with the standard I (McCapra and Hysert, 1973) and 0.15-0.16 for decanal, dodecanal and tetradecanal with the standard III (Shimomura et al., 1972). Thus, the quantum yield of long-chain aldehydes in the bacterial bioluminescence reaction appears to be in the range of 0.10-0.16. 

Inhibitors. Many common enzyme inhibitors show little or no effect on the activity of Cypridina luciferase in the luminescence reaction (Tsuji et al., 1974). However, EDTA strongly inhibits the bioluminescence reaction, showing a peculiar relationship between the... 

Fig. 3.2.6 Chemical structures of compound F (6 the euphausiid luciferin), a product obtained when F was left standing at -20°C for 2 weeks (7), and the product of bioluminescence reaction (8).

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. 

Fig. 3.3.3 Effects of temperature on the activities of luciferase ( ) and the quantum yields of coelenterazine (o) in the Oplophorus bioluminescence reaction. The activity was measured with coelenterazine (4.5 pg) and luciferase (0.05 pg), and the quantum yields with coelenterazine (0.2 pg) and luciferase (200 pg), in 5 ml of 15 mM Tris-HC1 buffer, pH 8.3 (at 25°C), containing 50 mM NaCl. Coelenterazine was first added to the buffer solution at the designated temperature, then the luminescence reaction was started by a rapid injection of 0.1 ml of luciferase solution. Replotted from Shimomura et al., 1978, with permission from the American Chemical Society.

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. 

Coelenteramide and coelenterazine. The structure of AF-350 contains the same aminopyrazine skeleton as in Cypridina etioluciferin and oxyluciferin (Fig. 3.1.8), suggesting that the bioluminescence reaction of aequorin might resemble that of Cypridina luciferin. To investigate such a possibility, we prepared the reaction product of aequorin luminescence by adding Ca2+ to a solution of aequorin. The product solution (blue fluorescent) was made acidic, and extracted with... 

Based on the available knowledge on the chemiluminescence and bioluminescence reactions of various luciferins (firefly, Cypridina, Oplophorus and Renilla), the luminescence reaction of coelenterazine is considered to proceed as shown in Fig. 5.4 (p. 171). The reaction is initiated by the binding of O2 at the 2-position of the coelenterazine molecule, giving a peroxide. The peroxide then forms a four-membered ring dioxetanone, as in the case of the luminescence... 

The decomposition of dioxetanone may involve the chemically initiated electron-exchange luminescence (CIEEL) mechanism (McCapra, 1977 Koo et al., 1978). In the CIEEL mechanism, the singlet excited state amide anion is formed upon charge annihilation of the two radical species that are produced by the decomposition of dioxetanone. According to McCapra (1997), however, the mechanism has various shortfalls if it is applied to bioluminescence reactions. It should also be pointed out that the amide anion of coelenteramide can take various resonance structures involving the N-C-N-C-O linkage, even if it is not specifically mentioned. 

Fig. 6.1.3 Bioluminescence reaction of Latia and the hydrolysis of Latia luciferin.

Fig. 6.1.7 Effect of pH on the initial light intensity and total light of Latia bioluminescence reaction in the presence of the purple protein, in 50 mM sodium phosphate buffer solutions having various pH values at 25°C (Shimomura et al., 1966b).

Fig. 6.3.4 Luminescence spectrum of the Watasenia bioluminescence reaction measured with a crude extract of light organs that contain particulate matters, in chilled 0.1 M Tris-HCl buffer, pH 8.26, containing 1.5 mM ATP. From Tsuji, 2002, with permission from Elsevier.

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.

Fig. 7.3.4 Kinetic profiles of the Diplocardia bioluminescence reaction, when Diplocardia luciferase, H2O2, or Diplocardia luciferin was injected last. In each case, 0.1 ml of the last component was injected into 0.9 ml of the mixture of other components, to give the final concentrations Diplocardia luciferase, 0.1 unit/ml Diplocardia luciferin, 32 mM and H2O2, 32 mM, in 0.1 M potassium phosphate buffer, pH 7.5. From Rudie et al., 1981, with permission from the American Chemical Society.

Fig. 8.9 Possible mechanisms of the bioluminescence reaction of dinoflagellate luciferin, based on the results of the model study (Stojanovic and Kishi, 1994b Stojanovic, 1995). The luciferin might react with molecular oxygen to form the luciferin radical cation and superoxide radical anion (A), and the latter deproto-nates the radical cation at C.132 to form (B). The collapse of the radical pair might yield the excited state of the peroxide (C). Alternatively, luciferin might be directly oxygenated to give C, and C rearranges to give the excited state of the hydrate (D) by the CIEEL mechanism. Both C and D can be the light emitter.

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