One of the joys of summer evenings is watching fireflies light up the night sky. These twinkling bugs communicate with one another using flashes of light — a trick biologists have appropriated to convey readouts from their experiments. Various forms of the enzyme responsible for the fireflies' luminescence — luciferase — can produce slightly different colours, ranging from red to yellow to green. Elsewhere in this issue, Toru Nakatsu et al. (Nature 440, 372–376; 2006) report several X-ray crystal structures of luciferase from the Japanese Genji-botaru firefly (Luciola cruciata, pictured) that explain how small changes in this protein can change the colour of the emitted light.

Credit: S. KURIBAYASHI

To create bioluminescence, magnesium, adenosine triphosphate and a small molecule called luciferin react with molecular oxygen. This reaction, which is catalysed by luciferase, generates an electronically excited oxyluciferin species. The yellow-green sparks we see in the night sky are emitted when oxyluciferin relaxes from its excited state to the ground state, losing energy in the form of light.

Single amino-acid changes in the active site of luciferase can alter the colour of the light emitted by the protein, but the chemical mechanism involved has been a mystery. Now Nakatsu et al. have obtained a series of ‘snapshots’ of the reaction — luciferase bound to the reactants (adenosine triphosphate and magnesium); an analogue of one of the reaction intermediates (known as DLSA); and the products (oxyluciferin and adenosine monophosphate).

The structure of luciferin bound to the reactants and the structure of the products are similar, both possessing an active site that is ‘open’ to the environment surrounding the protein. But when the protein is bound to DLSA, the active site closes: DLSA is tightly packed against several amino-acid side-chains, including isoleucine 288. Analysis of the three structures revealed that the side-chains of isoleucine 288 and serine 286 rotate when DLSA is present, closing the active site and forming a pocket.

The authors thought that the conformation of these amino acids might affect the colour of the light discharged by luciferase. So they solved the X-ray crystal structure of DLSA bound to a mutant luciferase known to emit red light. Their theory was borne out, as the active site of the DLSA-bound mutant protein was open.

The authors propose that the colour of the emitted light depends on the formation of this compact microenvironment during the reaction. In the normal protein, the excited oxyluciferin is held tightly in this pocket, packed against the side-chain of isoleucine, and yellow–green light is emitted. However, if this rigid pocket does not form — for example, in proteins that have mutations at isoleucine 288 or serine 286 — the excited oxyluciferin loses some energy, possibly because it can move around a little, and the protein emits red light, which is lower in energy.

In further work, Nakatsu et al. found that many of the other firefly luciferases that emit yellow–green light have either isoleucine or the slightly smaller amino acid valine at position 288. So it seems likely that the movement of the 288 amino acid is a common mechanism for controlling the colour of luciferase bioluminescence.