Firefly luciferase is the light-emitting

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

responsible for the

bioluminescence Bioluminescence is the emission of light during a chemiluminescence reaction by living organisms. Bioluminescence occurs in multifarious organisms ranging from marine vertebrates and invertebrates, as well as in some Fungus, fungi, microorgani ...

fireflies The Lampyridae are a family of elateroid beetles with more than 2,000 described species, many of which are light-emitting. They are soft-bodied beetles commonly called fireflies, lightning bugs, or glowworms for their conspicuous production ...

and click beetles. The enzyme catalyses the

oxidation Redox ( , , reduction–oxidation or oxidation–reduction) is a type of chemical reaction in which the oxidation states of the reactants change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is ...

firefly luciferin Firefly luciferin (also known as beetle luciferin) is the luciferin, precursor of the light-emitting compound, used for the firefly ( Lampyridae), railroad worm ( Phengodidae), starworm ( Rhagophthalmidae), and click-beetle ( Pyrophorini) biolumi ...

, requiring

oxygen Oxygen is a chemical element; it has chemical symbol, symbol O and atomic number 8. It is a member of the chalcogen group (periodic table), group in the periodic table, a highly reactivity (chemistry), reactive nonmetal (chemistry), non ...

and ATP. Because of the requirement of ATP, firefly luciferases have been used extensively in biotechnology.

Mechanism of reaction

The chemical reaction catalyzed by firefly luciferase takes place in two steps: *

luciferin Luciferin () is a generic term for the light-emitting chemical compound, compound found in organisms that generate bioluminescence. Luciferins typically undergo an enzyme-catalyzed reaction with Oxygen, molecular oxygen. The resulting transforma ...

+ ATP → luciferyl adenylate + PP_i * luciferyl adenylate + O₂ → oxyluciferin + AMP + light Light is produced because the reaction forms oxyluciferin in an electronically

excited state In quantum mechanics Quantum mechanics is the fundamental physical Scientific theory, theory that describes the behavior of matter and of light; its unusual characteristics typically occur at and below the scale of atoms. Reprinted, Add ...

. The reaction releases a photon of light as oxyluciferin goes back to the

ground state The ground state of a quantum-mechanical system is its stationary state of lowest energy; the energy of the ground state is known as the zero-point energy of the system. An excited state is any state with energy greater than the ground state ...

. Luciferyl adenylate can additionally participate in a side reaction with O₂ to form

hydrogen peroxide Hydrogen peroxide is a chemical compound with the formula . In its pure form, it is a very pale blue liquid that is slightly more viscosity, viscous than Properties of water, water. It is used as an oxidizer, bleaching agent, and antiseptic, usua ...

and dehydroluciferyl-AMP. About 20% of the luciferyl adenylate intermediate is oxidized in this pathway. Firefly luciferase generates light from luciferin in a multistep process. First, D-luciferin is adenylated by MgATP to form luciferyl adenylate and pyrophosphate. After activation by ATP, luciferyl adenylate is oxidized by molecular oxygen to form a dioxetanone ring. A

decarboxylation Decarboxylation is a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO2). Usually, decarboxylation refers to a reaction of carboxylic acids, removing a carbon atom from a carbon chain. The reverse process, which is ...

reaction forms an excited state of oxyluciferin, which tautomerizes between the keto-enol form. The reaction finally emits light as oxyluciferin returns to the ground state.

Bifunctionality

Luciferase can function in two different pathways: a bioluminescence pathway and a CoA-ligase pathway. In both pathways, luciferase initially catalyzes an adenylation reaction with MgATP. However, in the CoA-ligase pathway, CoA can displace AMP to form luciferyl CoA. Fatty acyl-CoA synthetase similarly activates

fatty acid In chemistry, in particular in biochemistry, a fatty acid is a carboxylic acid with an aliphatic chain, which is either saturated and unsaturated compounds#Organic chemistry, saturated or unsaturated. Most naturally occurring fatty acids have an ...

s with ATP, followed by displacement of AMP with CoA. Because of their similar activities, luciferase is able to replace fatty acyl-CoA synthetase and convert long-chain fatty acids into fatty-acyl CoA for

beta oxidation In biochemistry and metabolism, beta oxidation (also β-oxidation) is the catabolic process by which fatty acid molecules are broken down in the cytosol in prokaryotes and in the mitochondria in eukaryotes to generate acetyl-CoA. Acetyl-CoA enter ...

Structure

The protein structure of firefly luciferase consists of 550 amino acids in two compact domains: the

N-terminal The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...

domain and the

C-terminal The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When t ...

domain. The N-terminal domain is composed of two β-sheets in an αβαβα structure and a

β barrel In protein structures, a beta barrel (β barrel) is a beta sheet (β sheet) composed of tandem repeats that twists and coils to form a closed toroidal structure in which the first strand is bonded to the last strand (hydrogen bond). Beta-strands i ...

. The two β-sheets stack on top of each other, with the β-barrel covering the end of the sheets. The C-terminal domain is connected to the N-terminal domain by a flexible hinge, which can separate the two domains. The

amino acid sequence Protein primary structure is the linear sequence of amino acids in a peptide or protein. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end. Protein biosynthe ...

s on the surface of the two domains facing each other are conserved in bacterial and firefly luciferase, thereby strongly suggesting that the

active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding s ...

is located in the cleft between the domains. During a reaction, luciferase has a

conformational change In biochemistry, a conformational change is a change in the shape of a macromolecule, often induced by environmental factors. A macromolecule is usually flexible and dynamic. Its shape can change in response to changes in its environment or othe ...

and goes into a "closed" form with the two domains coming together to enclose the substrate. This ensures that water is excluded from the reaction and does not

hydrolyze Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution, elimination, and solvation reactions in which water is the nucleophile. Biological hydrolysis ...

ATP or the electronically excited product. Protein Structure

Spectral differences in bioluminescence

Firefly luciferase bioluminescence color can vary between yellow-green (λ_max = 550 nm) to red (λ_max = 620). There are currently several different mechanisms describing how the structure of luciferase affects the

emission spectrum The emission spectrum of a chemical element or chemical compound is the Spectrum (physical sciences), spectrum of frequencies of electromagnetic radiation emitted due to electrons making a atomic electron transition, transition from a high energ ...

of the

photon A photon () is an elementary particle that is a quantum of the electromagnetic field, including electromagnetic radiation such as light and radio waves, and the force carrier for the electromagnetic force. Photons are massless particles that can ...

and effectively the color of light emitted. One mechanism proposes that the color of the emitted light depends on whether the product is in the keto or

enol In organic chemistry, enols are a type of functional group or intermediate in organic chemistry containing a group with the formula (R = many substituents). The term ''enol'' is an abbreviation of ''alkenol'', a portmanteau deriving from "-ene ...

form. The mechanism suggests that red light is emitted from the keto form of oxyluciferin, while green light is emitted from the enol form of oxyluciferin. However, 5,5-dimethyloxyluciferin emits green light even though it is constricted to the keto form because it cannot tautomerize. Another mechanism proposes that twisting the angle between

benzothiazole Benzothiazole, or more specifically 1,3-benzothiazole, is an aromatic heterocyclic compound with the chemical formula . It is colorless, slightly viscous liquid. Although the parent compound, benzothiazole is not widely used, many of its derivativ ...

and

thiazole Thiazole (), or 1,3-thiazole, is a 5-membered heterocyclic compound that contains both sulfur and nitrogen. The term 'thiazole' also refers to a large family of derivatives. Thiazole itself is a pale yellow liquid with a pyridine-like odor and the ...

rings in oxyluciferin determines the color of bioluminescence. This explanation proposes that a planar form with an angle of 0° between the two rings corresponds to a higher energy state and emits a higher-energy green light, whereas an angle of 90° puts the structure in a lower energy state and emits a lower-energy red light. The most recent explanation for the bioluminescence color examines the microenvironment of the excited oxyluciferin. Studies suggest that the interactions between the excited state product and nearby residues can force the oxyluciferin into an even higher energy form, which results in the emission of green light. For example, Arg 218 has

electrostatic interactions Electrostatics is a branch of physics that studies slow-moving or stationary electric charges. Since classical times, it has been known that some materials, such as amber, attract lightweight particles after rubbing. The Greek word (), meani ...

with other nearby residues, restricting oxyluciferin from tautomerizing to the enol form. Similarly, other results have indicated that the microenvironment of luciferase can force oxyluciferin into a more rigid, high-energy structure, forcing it to emit a high-energy green light.

Regulation

D-luciferin is the substrate for firefly luciferase's bioluminescence reaction, while L-luciferin is the substrate for luciferyl-CoA synthetase activity. Both reactions are inhibited by the substrate's enantiomer: L-luciferin and D-luciferin inhibit the bioluminescence pathway and the CoA-ligase pathway, respectively. This shows that luciferase can differentiate between the

isomers In chemistry, isomers are molecules or polyatomic ions with identical molecular formula – that is, the same number of atoms of each element – but distinct arrangements of atoms in space. ''Isomerism'' refers to the existence or possibili ...

of the luciferin structure. L-luciferin is able to emit a weak light even though it is a

competitive inhibitor Competitive inhibition is interruption of a chemical pathway owing to one chemical substance inhibiting the effect of another by competing with it for binding or bonding. Any metabolic or chemical messenger system can potentially be affected b ...

of D-luciferin and the bioluminescence pathway. Light is emitted because the CoA synthesis pathway can be converted to the bioluminescence reaction by hydrolyzing the final product via an

esterase In biochemistry, an esterase is a class of enzyme that splits esters into an acid and an alcohol in a chemical reaction with water called hydrolysis (and as such, it is a type of hydrolase). A wide range of different esterases exist that differ ...

back to D-luciferin. Luciferase activity is additionally inhibited by oxyluciferin and

allosterically In the fields of biochemistry and pharmacology an allosteric regulator (or allosteric modulator) is a substance that binds to a site on an enzyme or receptor distinct from the active site, resulting in a conformational change that alters the p ...

activated "Activated" is a song by English singer Cher Lloyd. It was released on 22 July 2016 through Vixen Records. The song was made available to stream exclusively on ''Rolling Stone'' a day before to release (on 21 July 2016). Background In an interv ...

by ATP. When ATP binds to the enzyme's two allosteric sites, luciferase's affinity to bind ATP in its active site increases.

Homology

Firefly luciferase is thought to be a homolog of long-chain fatty acyl-CoA synthetase because of its ability to synthesize luciferyl-CoA from CoA and dehydroluciferyl-AMP. Inouye tested this hypothesis in 2010 by expressing the cDNA of ''Photinus pyralis'' and ''Lychocoriolaus lateralis'' luciferses in ''E. coli'' through cold shock gene expression. The resulting enzymes were then exposed to long-chain fatty acids, short-chain fatty acids, amino acids, and imino acids. Unsurprisingly, Inouye found that the luciferases only showed adenylation activity when exposed to long-chain fatty acids. The gene product of ''CG6178'' in ''Drosophila'' was also found to have high amino acid sequence similarity with firefly luciferase. While it did show high adenyltation activity when exposed to long-chain fatty acids, there was no luminescence when exposed to oxygen and LH₂-AMP– further suggesting that luciferase emerged as a long-chain fatty acyl-CoA homolog due to gene duplication.

Evolution

Phylogenetic analyses performed by Zhang ''et al''. (2020) suggest that the luciferses of the

Lampyridae The Lampyridae are a family of elateroid beetles with more than 2,000 described species, many of which are light-emitting. They are soft-bodied beetles commonly called fireflies, lightning bugs, or glowworms for their conspicuous production ...

, Rhagopthalmidae, and Phenogodidae families diverged from the

Elateridae Elateridae or click beetles (or "typical click beetles" to distinguish them from the related families Cerophytidae and Eucnemidae, which are also capable of clicking) are a family of beetles. Other names include elaters, snapping beetles, sp ...

family 205 Mya. According to phylogenetic data, the emergences of these two luciferases appeared even before the families could diverge– indicating their analogous nature due phenotypic convergences.

References

{{Enzymes Protein domains Oxidoreductases Bioluminescence Enzymes of known structure