NAD + + H 2 O C (+1) 2 e -

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1 Bioluminescence Bioluminescence is the biochemical emission of visible light by living organisms. As such, it is a special category of chemiluminescence. (Do not confuse bioluminescence with phosphorescence, which is a special form of fluorescence.) The few known bioluminescent organisms are scattered among diverse taxa in 2 of the 3 domains. (There are currently no reports of bioluminescence among the Archaea). Apparently bioluminescence has evolved independently at least several dozen times in lineages as diverse as bacteria, fungi, dinoflaggelates, insects, fish, and various other invertebrate phyla. Additionally, some nematodes, squid, and several groups of fish have evolved symbiotic relationships with bioluminescent bacteria. The preeminent bioluminescent Bacteria are Gram-negative marine bacteria in the genera Vibrio and Photobacterium. These genera are fairly closely related to each other, as are the biochemical systems responsible for light production. This, may reflect a single evolutionary origin of bioluminescence among Bacteria followed by horizontal gene transfer. Biochemical Mechanism Enzymes that catalyze light-emitting reactions in bioluminescent systems are called "luciferases". The organic co-substates for the luciferases are called "luciferins". Despite considerable diversity in luciferins (consistent with independent evolution of bioluminescent systems) all luciferases are flavin-dependent monooxygenase enzymes. BATEIAL Bacterial luciferase is a heterodimeric flavin-dependent monooxygenase that catalyzes the oxidation of an aliphatic aldehyde, dodecanal (bacterrial luciferin). The reaction mechanism, interestingly, is not very unique, being shared by other flavin-dependent monooxygenases (flavin-linked hydroxylases) which produce insignificant light emission. 3 Let's back up to consider the global context of this reaction in biochemical evolution. Most aldehyde oxidations involve NAD-linked dehydrogenase reactions in which 2 is not a substrate. The 2 electrons released by the oxidation of the aldehyde are transferred to NAD +, while the oxygen atom added to form the carboxyl group is derived from water. Typical Aldehyde xidation by NAD-dependent Dehydrogenase (+1) NAD dehydrogenase 2 e - (+3) 1 NAD + +

2 The corresponding reaction by a flavin-linked monooxygenase is: (+1) FMN oxygenase 4 e - (+3) FMN + 2 In this case the oxygen added to the substrate comes from 2, and water is a product, not a reactant. 2 of the 4 electrons needed to reduce the 2 are derived from the aldehyde oxidation, while the other two are donated by the flavin. Note that in the dehydrogenase reaction NAD + is electron acceptor. In the case of the oxygenase, FMN2 is electron donor. Various oxygenases of this ilk are known which do not emit significant light. The NAD-linked dehydrogenases are probably more ancient than the flavin-linked monooxygenases. The light emitting intermediate is an excited form of the Flavin. All bacterial luciferases emit blue light, the color which is least absorbed in sea water. owever, bioluminesence of some bacterial strains is yellow or green due to the presence of a fluorescent secondary protein. Bacterial Symbioses Some bioluminescent bacteria are symbionts (facultative or obligate) of marine fish or squid. In most fish-bacterium symbioses (10 families of 5 orders), the bioluminescent bacteria are found in some part of the gut and are similar to free living, culturable, Vibrio or Photobacterium species. Non-culturable P. fischeri (=V. fischeri) P. leiognathi Not identified P. phosphoreum Light organ symbioses in Anglerfish and Flashlight fish are obligate; the bacteria have not been cultured outside their hosts. P-based 16SrNA sequencing of the light organ symbionts show they are pure cultures of a single symbiont strain derived from the same common ancestor as the facultative Photobacterium symbionts and free-living marine Vibrios i. Anglerfish Suborder eratioidei in 9 of 11 families females have luminescent lure; deep-sea predators Flashlight fish Family Anomalopidae shallow-water plankton feeders; large suborbital light organs for conspecific communication 2

3 FIEFLY The bioluminescent reaction in fireflies involves adenylation of the luciferin. GEEN FLUESENT PTEIN GFP shifts the blue light emission of Aequorin (466 nm) to green (508 nm). GFP is a 11-stranded β-barrel protein threaded by an α-helix running up the axis of the internal cylinder. The chromophore responsible for fluorescence is attached to the α-helix and is buried almost perfectly in the center of the cylinder. Aequoria (Jellyfish) The bioluminescent reaction in Aequoria is a-dependent. Note the complexity of the nidarian luciferin coelenterazine.) Green Fluorescent Protein GFP shifts the blue light emission of Aequorin (466 nm) to green (508 nm). GFP is a 11-stranded β-barrel protein threaded by an α-helix running up the axis of the internal cylinder. The chromophore responsible for fluorescence is attached to the α-helix and is buried almost perfectly in the center of the cylinder. The fluorescent chromophore is a 4-(phydroxybenzylidene)imidazolidin-5-quinolone attached to the peptide backbone through the 1- and 2-positions of the ring. 3

4 The chromophore is formed spontaneously from residues Ser65-Tyr66-Gly67 in the native protein. Thus the GFP gene contains all the information necessary for the posttranslational synthesis of the chromophore, no additional enzymes are needed. Applications Bacterial lux genes have been cloned and used as "reporter genes" ii. Light emission can be easily detected and measured by simple electronic luminometers. Bioluminescent bacteria are the basis of a general toxicity assay iii. Instrumentation and reagents are marketed commercially by as the Microtox system by Microbics orp., arlsbad, A. Firefly luciferin/luciferase is used as a sensitive assay for ATP. Jellyfish (Aequoria) luciferinliciferase is used as a sensitive assay for a ++. GFP of jellyfish (Aequoria) is an important fluorescent tag and reporter gene product. The luminous organ of a marine fish is used by at least one cultural group as a lure for fish. 4

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