Fireflies communicate with one another with flashes of light. Investigators have long been intrigued by this process, especially because the insects emit the light without generating heat. Although studies have established that the bioluminescence reaction is facilitated by the enzyme luciferase, the precise mechanism has proved elusive. Researchers at Riken Harima Institute in Hyogo and at Kyoto University, both in Japan, addressed the problem in a recent Nature letter. “I wanted to know … the mechanism [by which] luciferase prevents the ‘waste’ of the chemical energy and converts the energy into light so efficiently,” said Hiroaki Kato, principal investigator of the study. A previous study revealed that single amino acid changes in the Japanese firefly’s luciferase can produce changes in the color of the light emitted, which suggests that the key to the color control mechanism (and therefore to the light-emitting mechanism) can be found in the dynamic structural changes of luciferase during the bioluminescence reaction. For this reason, Kato and colleagues used x-ray crystallography to determine the three-dimensional structures during three stages: a resting state prior to the reaction; an excited state just before light is emitted; and a second resting state after the reaction. Researchers have uncovered the mechanism that allows fireflies to emit light without generating heat. The enzyme responsible for the bioluminescence has an “open” structure before and after the light is emitted. During catalysis, however, it adopts a “closed” form, which minimizes the loss of energy. Reprinted from Nature with permission of the researchers. He noted that it is very difficult to observe the “true” excited state because this high-energy state is unstable and short-lived. He and the others overcame this problem by using a stable analogue of a high-energy intermediate as a ligand. The ligand “tricks” the enzyme into catalyzing the reaction, allowing the researchers to see the unique structure present only in the excited state. This allowed them to determine the mechanism underlying the “cold light” of firefly bioluminescence. They found that the active site of luciferase binds the excited state very tightly in a highly rigid environment, preventing the chemical energy of the excited state from escaping as heat. The energy is instead released as light with a high quantum yield. The study followed a previous one that had shown that single amino-acid changes in the firefly’s luciferase enzyme can produce changes of color: from yellow-green to red, for example. This suggests that the key to the mechanism underlying the bioluminescence reaction could be found in the structural changes of luciferase during the reaction. These findings may have important implications. For example, advanced understandings of the structure-function relationships that are involved in the bioluminescence reaction could result in improvements in bioluminescence imaging based on in vivo expression of firefly luciferase, which researchers are already using for noninvasive detection of tumors in mice. Such applications would benefit from modified luciferase exhibiting more brilliant and continuous light. “Our findings should enable researchers to design such a superluciferase,” Kato said. In the meantime, the investigators are planning to design mutant luciferase capable of emitting other colors — especially blue — which would offer a still higher quantum yield. “The quantum yield of the luciferase reaction is already about 90 percent,” Kato said. “How do we increase the energy of the excitation state? It seems impossible. That’s why I’m trying to do it.” Nature, March 16, 2006, pp. 372-376.