Osamu Shimomura

There are numerous kinds of luminous organism on earth. Mysterious emission of light from them inspired the curiosity of mankind ever since the ancient times. In history, Raphael Dubois discovered luciferin and luciferase from one of them, a click beetle, in 1885

I'd like to talk about Chemistry, chemical mechanisms by which bioluminescent organisms emit light. There are great many kinds of bioluminescent organisms among us, but most of them live in the sea. Only several kinds are listed here on land and rest of hundreds, these are all in the sea. In luminous organisms light is produced by chemical reactions and those reactions can be categorized into three broad types, luciferin-luciferase type and photoprotein type and other type. Luciferin is a generic name of the compound that in its right will oxidize in the presence of enzyme luciferase. An important criterion of luciferin-luciferase reaction is that the amount of light emitted is proportional to the amount of luciferin. In photoprotein type luciferin is incorporated in the molecule of a protein and no other protein or enzyme is needed for light emission. The amount of light emitted is proportional to the amount of the photoprotein reacted. Other types include the bioluminescence systems that do not involve any luciferase or protein. At the moment we have only one example, the luminous fungus that belongs to other type. Today we have substantial chemical understanding with nine kinds of bioluminescence systems that is only nine kinds from hundreds of different kinds of bioluminescence organisms. Several of them are luciferin-luciferase type. And the two are photoprotein type. I would like to briefly explain the nine bioluminescence reactions. The first is firefly. Firefly is so popular and well-known, sometimes used in postal stamps. The Luciferin of a firefly is a thiazole compound. Luciferin has a one asymmetric carbon and only D-form is active in light emission. When luciferin is oxidized in the presence of luciferase and ATP oxyluciferin is produced and accompanied by light emission. This is the mechanism of the reaction. Luciferin first reacts with the ATP and product Adenylate, this is oxidated with carbon and other forms of hydroperoxide... which instantly decomposes into oxyluciferin accompanied by light emission. In acidic media red light is emitted. In alkaline media yellow-green light is emitted. The structure of the light emitter, when considered initially, this forms red light and this forms yellow-green light but doubt was raised around 1990 of this structure. In the year 2004 Branchini et al. reported that luciferin contained two major groups at this position, that cannot energize like this, can emit yellow-green light, so this structure is wrong. Now it is believed that red light is emitted from this structure and yellow-green light is emitted from this structure. Firefly bioluminescence is very essential to ATP and widely used in the assay of ATP. Next is coelenterate luciferase reaction is very widely distributed in many organisms. It is probably the most important compound in bioluminescence. For example... see feather, some jellyfish... This jellyfish is an interesting one. This periphylia is distributed very widely in the world but in certain fjords in Norway it grows to a very large size. This only is about 800g and estimated to be about twenty-five years old. Coelenterate luciferin is also in many kinds of luminous shrimp, deep sea fish and squids. This is a structure of coelenterazine that is the emitting compound. Coelenterazine is oxidized in the presence of luciferin in the coelenteramide, accompanied by light emission. The mechanism involved is this. This position is first oxygenated giving hydroperoxide. which recomposes into coelenteramide and CO2 accompanied by light emission. Next example of luciferin-luciferase reaction is this cypirdina. Cypirdina is a tiny coruscation very small but it emits very strong blue light. The luciferin of a cypirdina is also emitted in compound with three substances, these all different from coelenterazine. The reaction mechanism is almost the same with that of coelenterazine: First forming of hydroperoxide, and then it decomposes into oyxluciferin and etioluciferin and CO2 accompanied by light emission. Next is luminescence of bacteria, this picture shows my former boss Frank Johnson admiring the culture of luminous bacteria. The luciferin of luminescence bacteria is a long-chain fatty aldehyde. In this case tetradecanal. And luciferin is oxidized in the presence of FMNH2 into the corresponding fatty acid accompanied by light emission. This is the mechanism involved. First FMNH2 is oxygenated at this position forming a hydroperoxide which reactions aldehyde and forms a peroxide. When this peroxide decomposes, hydro FMN is formed and hydroxide FMN is in an excited state and where an excited state light is emitted. Peroxide FMN decomposes into FMN plus water. This is a tiny latia found in streams of New Zealand, this produces slime which emits bioluminescence. Here we are collecting latia, luciferin of latia is a sesquiterpene. It has a functional group of enol formate of an aldehyde, very unstable in water. Luciferin is oxidized through this structure into this ketone, plus 2 formic acidic accompanied by light emission. In the case of the luciferin is a very simple compound, an aldehyde. This aldehyde forms a duct with hydroperoxide giving this, and with this duct they compose into acid accompanied by light emission. This is the last example of luciferin-luciferase reaction. This is a Krill, very small shrimp about 2~3cm long and they have a ten photo foot and at the base of the legs and they emit very strong pinpoint light. The luciferin of Krill is almost identical to luciferin with the luciferin of dinoflagellate, both luciferin have tetrapyrrole structure. The only difference is this X group. In Krill X=OH and in dinoflagellate X = H. Luciferin is oxidized at this position. In the presence of luciferase and light is emitted. I should note that Krill is the most abandoned animal on earth. Next is a photoprotein type luminescence. This is Aequorea. Aequorea emits green light at the edge of the umbrella. It is green light. Aequorea contains a photoprotein namely Aequorin. Aequorin is a protein the molecular weight of about 20,000. And contains a functional group of coelentarazine peroixde at the centre of the molecule. If calcium exists calcium binds through the outside of the protein and causes deformation of protein which triggers the composition of coelenterazine peroxide into coelenteramide and CO2 accompanied by light emission. The light is blue. The protein part produced after removing the culture can be put back, degenerated into original decoding by integration with coelentarazine and oxygen. If molecule green GFP exists near the molecule Aequorin the energy produced by Aequorin with reaction with the culture is transferred to the molecule GFP and the GFP emits green light instead of the blue light from Aequorin. Aequorin and GFP are both very useful and widely used in research. Another example of photoprotein is contained in this squid, symplectoteuthis. This squid contains photoprotein named Symplectin. Symplectin has a molecule of coelenterazine which is spontaneously oxidized in coelentaramide accompanied by light emission. In the squid Symplectoteuthis, Symplectin is formed by Aequorin and the hydrocoelenterazine. Now, I'd like to talk about luminous fungus. About forty species of luminous fungus are known. And all of them emit yellowish light with emission rate of 530nm. This is mycena mushroom, in the case of luminous fungi, luminescence is continuously and steadily emitted for a long time usually several days. This is another luminous fungus, panellus. This mushroom is interesting. If the mushroom is dryed in dry weather the luminescence stops but when it gets wet by rain luminescence starts again. Panellus contains a high content of luciferin precursor compound. This precursor reacts with methenamine in the presence of GFP and luciferin. Luciferin is made of three molecules of precursor and the two molecule of methenamine. The structure is not completely known but according to modern study the structure might be like this. This luciferin emits light in the presence of superoxide, oxygen and tetradecanoylchlorine. This is a cationic detergent or maybe sulfoxide. The mushroom luminescence can take place in the absence of any protein or luciferase. The role of luciferase is replaced by the action of cationic sulfoxide. By the way tetradecanoylchlorine exists in mushrooms and many animals. Most of the information I obtained before 1980, in recent years there are many researchers working on the application of bioluminescence and the application is flourishing. But only a few researchers are working on the fundamental aspects of bioluminescence. And as far as I know nobody is working to try to find the structure and the chemistry of a new luciferin. Although there are many luciferins left to be studied. Considering that luciferins are the most fundamental elements in bioluminescence and also that luminous organism is sort of a treasure box containing interesting new compounds and reactions judged by past results, I feel sorry about the present status. Studying the new bioluminescence system may not be easy, mainly because there is no protocol to use you have to develop the protocol by yourself for each new system. I believe the work is for young energetic, gifted people, it is a challenging work and I'm hoping that some of you are interested in the chemistry of bioluminescence. Thank you.