What is bioluminescence?

Bioluminescence is frequently defined as light emission from living organisms based upon a chemiluminescent reaction which is catalysed by a specific enzyme. The term was probably first employed by Harvey in 1916 but since then much confusion has surrounded terms and definitions within the field of luminescence.

Luminescence is the name given to all forms of light phenomena that do not result from a rise in temperature. Luminescence reactions can be either phosphorescent or fluorescent. The term phosphorescence is defined as light emission caused by electronic transitions between levels of different spin multiplicities whereas the term fluorescence is applied to light emissions connected by electronic transitions between levels of like multiplicity. Phosphorescence is responsible for light persistence for up to several seconds whereas fluorescence is an almost instantaneous act, lasting only nanoseconds after excitation. This would by default place bioluminescence within the fluorescence category. However, fluorescence and bioluminescence are considered to be two separate processes and this is where the contradictions in definitions begin. Fluorescence is defined as being reliant upon photons as the energy source whereas bioluminescent reactions are dependent upon a chemical reaction for their energy source.

Bioluminescence in Nature

Bioluminescent organisms are found in almost all phyla and all the systems involved in making light share a basic biochemical pathway that are luciferase-catalyzed reactions of the molecular oxygen with a substrate generically known as a luciferin. The term luciferase is also generic as luciferases from different groups of organisms are unrelated genetically as well as structurally. All bioluminescent reactions include a luciferase-bound peroxy-luciferin intermediate that breaks down to provide energy for excitation. Light is generated in a bioluminescent reaction very rapidly as a part of a multi-step chemical reaction. The penultimate step is the production of a molecule in an electrically excited state P* that has a very short lifetime, 1-10 nanoseconds. Bioluminescent organisms range in diversity from bacteria, fungi, and algae through to earthworms, squid and fish and occur in 700 different genera.

The Bioluminescent Reaction

The first modern study of bioluminescence began with a luminous click beetle. In 1885 Dubois demonstrated the first example of a luciferin-luciferase reaction from a West Indies Pyrophorus species by preparing two extracts from the light organs that, when combined, produced light [6]. Dubois concluded that one extract contained a heat labile enzyme necessary for the light emission and called this ‘luciferase’ whilst the other extract contained a heat stable substance he designated “luciférine”. The requirement of adenosine tri-phosphate (ATP) and Mg2+ for the bioluminescent reaction were identified in the 1940’s and in 1978 McElroy & DeLuca proposed a two step scheme for the overall reaction of firefly bioluminescence.

Both steps are catalysed by the enzyme luciferase (E). In the first stage luciferin (LH2) is converted into a luciferyl adenylate (LH2-AMP) by ATP in the presence of Mg2+. In the second step, luciferyl adenylate is oxidized by molecular oxygen resulting in the emission of light and the production of oxyluciferin (L). Luciferin is a general term defined as an organic compound that exists in a luminous organism and provides the energy for light emission by being oxidized, normally in the presence of a specific luciferase. Firefly luciferin was first purified and crystallized in 1957 ultimately leading to the determination of its structure in 1961. The product of the luminescent oxidation of luciferin is oxyluciferin, a compound which is extremely unstable . A number of studies focused on the intervening steps between luciferin and oxyluciferin resulting in the postulation of the bioluminescent reaction shown in figure 3. Luciferase-bound luciferin is converted into an adenylate in the presence of ATP and Mg2+ with the release of pyrophosphate (PPi). The adenylate in the presence of oxygen forms a peroxide intermediate (A) which then forms a dioxetanone intermediate (B) by splitting off AMP. Dioxetanes are heterocyclic compounds which consist of a four-membered ring that contains two oxygen atoms and two carbon atoms. The subsequent decomposition of the dioxetane intermediate produces an excited state of oxyluciferin in the form of either a monoanion (C1) or a dianion (C2). When the energy levels of the excited states fall to the ground states, C1 emits red light ( 615nm) and C2 emits yellow-green light ( 560nm).

Beetle Luciferase
The luciferase from the firefly Photinus pyralis was first purified, crystallized and partially characterized in 1956. The molecular weight was estimated as 100,000 and the isoelectric point at pH 6.2-6.3. In 1984 Wood and co-workers cloned P. pyralis luciferase by in vitro translation and determined the molecular weight to be 62,000 as opposed to the previously reported 100,000. Wienhausen and DeLuca identified luciferases from other bioluminescent beetle species, including the click beetle Pyrophorus plagiophthalamus. These migrated at a similar position, although not identical, and exhibited extensive cross-reactivity with antibodies raised against P. pyralis luciferase. Thus it was anticipated that luciferases from other bioluminescent beetles would have similar molecular weights.

The firefly P. pyralis was again used to provide the material for the first cloning of luciferase into a bacterial system. De Wet and coworkers in 1985 expressed the cDNA of P. pyralis luciferase in Escherichia coli providing the basis for mass production of luciferase in vitro and the further characterization of the enzyme through mutagenesis studies in the coming years. To date the luciferase cDNA has been characterized from over twenty bioluminescent beetle taxa and extensive information has been collated about these enzymes. In fireflies the luciferase enzyme is composed of one polypeptide chain ranging in size from 545–552 highly conserved residues. Over half are non-polar or ambivalent amino acids and the number of charged residues is virtually the same for all lampyrid species.
It was not until 1996 that Conti et al. resolved the crystal structure of the P. pyralis luciferase at a resolution of 2.0 Å. The protein was found to be folded into two compact domains connected by a short flexible hinge . The large N-terminal domain being composed of a ß-barrel and two ß-sheets flanked by a-helices to form an aßaßa five-layered structure. The C-terminal portion of the molecule formed a distinct domain separated from the N-terminal domain by a wide cleft. Conti et al. proposed that the cleft was far too big to accommodate the substrate and the domains will close in the course of the reaction to sandwich the substrates.
The bioluminescent mechanism in the Phengodidae and luminescent members of the Elateridae is considered to be the same as that found in the fireflies (Lampyridae). Each mechanism is dependent upon ATP, luciferin, Mg2+ and the enzyme luciferase to create light. Beetle luciferin is regarded to be the same structure in the Phengodidae and Elateridae as the Lampyridae. Despite these similarities the difference in colours of light produced in these families is quite dramatic. In lampyrids the light is limited in range from green to yellow (538–584nm). However, bioluminescent click beetles have three light organs; a pair of dorsal oval light organs on the pronotum which emit a green light ( 536–559nm) and a ventral organ located on the first abdominal segment which ranges in colour from green through to orange ( 549 – 594 nm). In railroad worms the number of lanterns increases with eleven pairs of luminous organs located dorso-laterally along the abdominal and thoracic segments. These emit green through to orange light ( 535 – 592 nm) and are present in both adults and larvae. In addition, some species such as the railroad worm Phrixothrix have a luminous organ on the head which emits red light ( 600–620nm). These colour differences are a result of amino acid differences in the luciferase protein.