Bio Chemistry in Fireflies - Bioluminescence

Enlighten Your Understanding of Bioluminescence in Fireflies

There's no question Owl City's song Firefly is a bop, but have you ever wondered how the ten million lightning bugs lit up the sky? (That was my attempt at a shoddy introduction. But, if you were really wondering... the song is a metaphor, dingus. I'd like to see ten million lightning bugs teach you how to dance.) Well, the mesmerizing, blinking lights on fireflies are produced by a process called bioluminescence. In this process living organisms convert chemical energy into light. The light comes from the oxidation of an organic substrate, a luciferin, catalyzed by an enzyme called a luciferase. There are so many different organisms that emit light in nature including bacteria, fungi, crustaceans, mollusks, fishes, and insects. While all of the specific biochemistries involved in these different cases of bioluminescence are diverse, all include an enzyme-mediated reaction between molecular oxygen and an organic substrate. Also, most, if not all, bioluminescence processes involve the formation and breakdown of a four-member ring peroxide or a linear hydroperoxide. While bioluminescent fish really just have symbiotic relationships with bioluminescent bacteria, fireflies produce chemical reactions inside their bodies that allow them to light up.
There are three families of luminous beetles that represent about 2,000 species. These families are Lampyridae (true fireflies), Phengodids (click beetles), and Elateridae (glow-worms). Recent studies of the key structure-function relationships that account for the efficient enzyme-catalyzed emission of light in the firefly give us a better understanding of how exactly light is produced by fireflies. Applications of these studies include medical and pharmaceutical methods like in vivo imaging to visualize tumors and monitoring gene expression and regulation.

Biochemical Reactions

Firefly bioluminescence requires multiple biochemical reactions represented below. Luc represents firefly luciferase which is a euglobulin protein that catalyzes the oxygenation of luciferin using ATP (the universal biochemical energy source adenosine triphosphate) and molecular oxygen to yield oxyluciferin, a highly unstable, singlet-excited compound that emits light upon relaxation to its ground state. PP_i is inorganic pyrophosphate. In the first step, luciferase converts firefly D-luciferin (LH₂) into the corresponding enzyme-bound luciferin adenylate. In the presence of luciferase D-LH₂-AMP synthetically reacts with oxygen to produce light emission identical to light obtained with natural substrates D-luciferin and Mg-ATP.
Even though it does so without the involvement of a metal or cofactor, the luciferase enzyme functions as a mono-oxygenase. Scientists are still trying to determine how luciferase amino acid residues are recruited to promote the addition of molecular oxygen to the D-luciferin adenylate. The D-luciferin adenylate is then transformed to an electronically excited state oxyluciferin molecule and carbon dioxide (each contains one oxygen atom from molecular oxygen). The rapid loss of energy of the excited state oxyluciferin molecule via a fluorescence pathway results in visible light emission. The high quantum yield of 0.4-0.6 for the emission of a photon from a reacted LH₂ molecule reflects both efficient catalytic machinery and a highly favorable environment that prevents electronically excited state energy loss by nonlight-emitting pathways. 

Mechanism of Bioluminescence

After the formation of enzyme-bound luciferyl adenylate (step a), from the C-4 carbon of the adenylate, a proton is abstracted by a basic side chain amino acid residue of luciferase (step b). Then, molecular oxygen adds to the newly formed anion (step c) and a highly reactive diozetanone intermediate produces an electronically excited state oxyluciferin molecule and carbon dioxide (step e). Chemiluminescence model studies, red light emission, which is observed at pH 6.0, results from the keno form of the emitter. The neonate assisted tautomerization (step f) produces the yellow-green light emission, which is observed at pH 8. However, recent experimental studies with a firefly luciferin analog have shown evidence consistent with the keno form of oxyluciferin alone being capable of producing all of the colors of firefly bioluminescence. Beetle luciferases in nature display different colors of light, ranging from green to red. Scientists are still reaching conclusions, but perhaps luciferase modulates emission color by altering the resonance-based charge delocalization of the excited state, shown below. 

The Light Organ

Unlike a light bulb, a firefly's light does not produce a lot of heat in addition to light. Fireflies produce "cold light" which is the emission of light without a lot of energy being lost as heat. Fireflies also have a light organ. This organ controls the beginning and end of the chemical reaction explained above, and thus the start and stop of its light emission by adding oxygen to the other chemicals needed to produce light. However, insects don't actually have lungs and instead transport oxygen through a complex series of successively smaller tubes known as tracheoles. The process of the tracheoles is slow due to the slow moving muscles that control oxygen transport and this created a mystery as to how some firefly species manage such a high flash rate. Fairly recently researchers learned that nitric oxide gas (also known as laughing gas) plays a critical role in firefly flash control. When the firefly's light is "off" no nitric oxide is being produced. This means that oxygen that enters the light organ is bound to the surface of the cell's mitochondria, and is not able to go further within the light organ. However, when nitric oxide binds to the mitochondria it allows oxygen to flow into the light organ where it combines with the other chemicals needed to produce the bioluminescent reaction. The flashes of light are fairly quick because nitric oxide breaks down very quickly and once it's gone the oxygen molecules are trapped by the mitochondria again, meaning they cannot contribute tot he production of light.

Reasons Fireflies Light Up

Fireflies often produce defensive steroids in their bodies that make them unappealing to predators. Larvae can glow as well and use them as warning displays to communicate their distastefulness. Also, many different species of fireflies have different flash patterns and use them to identify members of the opposite sex. Studies have even shown female fireflies are turned on (get it?) by specific male flash pattern characteristics such as higher flash rates and increased flash intensity.

Work Cited:

https://www.scientificamerican.com/article/how-and-why-do-fireflies/ 

http://photobiology.info/Branchini2.html