Firefly Luciferase

Luciferase Mechanism

Firefly luciferase is a euglobulin protein that catalyses the oxygenation of luciferin using ATP and molecular oxygen to yield oxyluciferin, a highly unstable, singlet-excited compound that emits light upon relaxation to its ground state:

Luciferase Reaction

The reaction proceeds in two parts: the adenylation of luciferin, followed by the oxygenation of adenyl-luciferin. The adenylation step activates luciferin as an enzyme-adenyl-luciferin complex, which is analogous to the activation of fatty acids by acyl-CoA ligases (based on homology, beetle luciferases are thought to have evolved from acyl-CoA ligases, retaining their catalytic mechanism for adenylation). In the second step, luciferase acts as an oxygenase on adenyl-luciferin to produce oxyluciferin and carbon dioxide, the decay of oxyluciferin producing a photon of light.

Fireflies at night

Colour Variation

Subtle variations in the structure of beetle luciferases can produce different coloured luminescence. Luciferase must undergo a considerable conformational change prior to the oxidation step in order to prepare a hydrophobic environment ready for the unstable oxyluciferin, thereby minimising energy loss. Changes in structure that affect the ability of the active site to exclude water can alter the colour of bioluminescence.

In addition, the type of residue at position 288 in the luciferase protein directly affects colour emission. This residue must be hydrophobic, but the size of its hydrophobic side chain can alter the colour of light emitted. Most fireflies emit yellow-green light and have an isoleucine or leucine residue at position 288, while luciferases that emit a different colour have a different residue at this position.

Luciferase, Lighting up Experiments

Purified firefly luciferase enzyme has widespread uses in scientific and medical research. Bioluminescent imaging (BLI) is a sensitive and versatile tool that uses luciferase enzyme as an imaging reporter. It has been used to track tumour cells, bacterial and viral infections, gene expression, signal transduction, cell trafficking and the cellular response to treatment in whole organisms. Using luciferase as an imaging reporter for cancer provides insight into cancer-specific molecular mechanisms within the context of the whole animal. In addition, firefly luciferase has been used to locate primary tumours and their metastases in animal studies.

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