Guiding light: the mysteries of firefly bioluminescence unfold

In 2011, HFSP awarded a Young Investigator Grant to an international team of researchers to study the "Excited-State Structure of the Emitter and Color-Tuning Mechanism of the Firefly Bioluminescence".  The findings of this successful four-way collaboration between the labs of Pance Naumov (Japan/Abu Dhabi), Pascal Didier (France), Lukas Hintermann (Germany) and Michel Sliwa (France) are described in the following article by Pance Naumov, Principal Investigator on the grant team.      

After acquiring his Ph.D. in chemistry and materials science from the Tokyo Institute of Technology in 2004, Pance Naumov continued his research as an independent research fellow of the National Institute for Materials Science in Japan. There he established a new laboratory for the study of solid phenomena, while simultaneously performing research at two Japanese synchrotrons. In 2007, he was appointed Associate Professor at Osaka University, where he established a second, new laboratory for solid-state chemistry, and led a small but very active research group and in 2012 he became an Associate Professor at New York University’s new campus in Abu Dhabi (NYUAD).

Link to the Naumov group

Pascal Didier is a French Assistant Professor at the University of Strasbourg. He received his Ph.D. in Physical Sciences under the supervision of J-Y. Bigot. His thesis was entitled “Optical properties of fluorescent protein studied by femtosecond spectroscopy”. After his Ph.D., he joined the group of Pr. B. von Issendorf at the University of Freiburg in Germany for one year to study the electron dynamics in metallic clusters by using pump-probe spectroscopy. In 2006, he was recruited as an Assistant Professor and is now working at the University of Strasbourg in the Laboratory of Biophotonics and Pharmacology. His main research is focused on understanding biomolecular interaction in living cells by using quantitative fluorescence microscopy. He is involved on a broad range of topics such as single molecule experiments, high-resolution fluorescence microscopy, and optical characterization of organic nanoparticles… He has published > 35 articles in international journals.

Link to Didier group

Lukas Hintermann was born in Switzerland in 1972. He studied chemistry at ETH Zurich and was awarded a Ph.D. in 2000 for work on asymmetric catalytic fluorination performed with Antonio Togni. In 2001– 2002 he moved to the Tokyo Institute of Technology as JSPS postdoctoral fellow in the group of Keisuke Suzuki. He then started independent research projects at RWTH Aachen University in Germany, hosted by Carsten Bolm and supported by an Emmy Noether program of Detusche Forschungsgemeinschaft (DFG). In 2009, he became Associate Professor at the Technische Universität München (TUM).

Link to the Hintermann group

Michel Sliwa is a French CNRS researcher and a former student from the Ecole Normale Supérieure de Cachan where he received his Ph.D. in Physical Sciences under Pr. K. Nakatani on SHG photo-switching and ultrafast spectroscopy. After his Ph.D., he joined the group of Pr. J. Hofkens at the Katholieke Universitat Leuven for two years to gain knowledge on single-molecule fluorescence spectroscopy. In 2007, he was recruited as a CNRS researcher and is now working at the University Lille Nord de France in LASIR. His main research is focused on understanding the ultrafast photodynamic of new photo-active (bio-) systems using ultrafast and single molecule spectroscopies. He has published > 75 articles in international journals and in 2013 was awarded the CNRS Bronze Medal which acknowledges young researcher's work.

Link to the Sliwa group (in French)

Light is ethereal, but eternal. The generation of cold light by living organisms, known as bioluminescence, has fascinated and puzzled mankind for centuries. The reasons behind the exceedingly complex spectrochemistry of oxyluciferin, the emitting species in firefly bioluminescence, were finally revealed.

The mesmerizing flashes of fireflies, a striking example of bioluminescence, continue to inspire laymen, artists, writers and Hollywood directors. The generation of light is essential to the survival of a plethora of organisms that produce and utilize light for defense, attraction, or mating. Bioluminescence is a more common phenomenon than was initially believed; it has been reported in more than 70 biological families, spanning over 250 genera. As an illustration of the diversity in bioluminescence, one family only of Lampyridae, more commonly known as fireflies, contains over 5 genera and about 2000 species. Even though the phenomena is a fascinating sight to behold, the basic research in bioluminescence is driven by practical applications. Bioluminescence has revolutionized microscopic techniques for imaging of live cells and tissues, and is now the basis for a number of modern bioanalytical methods that include detection of attomolar quantities of bacterial contamination of food. There are also other microscopy techniques based on imaging of living cells that utilize bioluminescent tags that are essential for diagnostics of a number of diseases, including cancer. The convenience of direct visualization of molecular and intracellular pro­cesses, their biochemical pathways and, more importantly, with monitoring of their evolution and spatial progression both in vivo and in vitro has led to the burgeoning research driven by the specific requirements related to diagnostics applications. The research in bioluminescence was boosted by the Nobel Prize in chemistry, which was awarded in 2008 for contributions to the basic research on this process.

  

Photo credit: Michel Sliwa

Aside from the fascination with the visual effect, at a molecular level bioluminescence is a mechanistically intriguing process. In living organisms, bioluminescence occurs within enzymes called luciferases. At the outset of the multistage biochemical reaction, a chemical system within the luciferase transitions from the ground (unexcited) state to energetically excited state by making use of the energy stored within the chemical bonds. This process, called chemiexcitation, proceeds by a mechanism that is very different from the more common process of excitation by light (photoexcitation). Similar to the fate of the photoexcited molecules, the chemiexcited molecules spontaneously return to the ground state by emitting light. This phenomenon of conversion of chemical energy directly into light is one of the most fundamental cases of energy transduction in Nature.

Yet, many of the fundamental aspects of bioluminescence remain enigmatic and unexplored, including, first and foremost, the chemical nature of the emitting species within the luminous systems of many organisms. In fireflies, the emitting molecule, called oxyluciferin (the name comes from the Latin for “Lucifer” meaning light bearer) can exist in up to as many as six possible chemical forms. The identity of the emissive species, which could change in the course of the chemiluminescent reaction, has not been established yet. A second major conundrum is the origin of the different color of light emitted by some natural and mutant luciferases. Even though the emitter molecule present in several organisms is identical, the emitted color depends on the species and can range from green to red; fireflies (Lampyridae) emit yellow-green light, click beetles (Elateridae) produce green to orange light, while railroad worms (Phengodidae) emit green to red light. This multicolor emission could be important for advanced microscopy techniques, since a limitation to most of the currently employed bioluminescent systems relative to fluorescent probes, is posed by their fixed wavelength of emission determined by properties of the substrate-luciferase complex. Many applications would profit from highly specific, but variable and complementary emission with intrinsically tunable wavelength. At the core of this color tuning is an understanding of the mechanism by which the fireflies, click beetles and railroad worms can emit light of different color, a mystery that has remained unresolved for centuries.

In many ways, the oxyluciferin is a very special molecule. The structure of the firefly oxyluciferin has long remained unobserved and it was believed that it was too unstable to be isolated and crystallized [1]. In 2009, we succeeded in crystallization and the first structure determination of firefly oxyluciferin [2], followed by preliminary spectroscopic analyses [3]. More recent detailed analysis of its chemistry clarified [4] that the early assumptions of its instability due to purported oxidation in air were incorrect; indeed, firefly oxyluciferin is stable in aerobic conditions at room temperature in neutral and in weakly acidic conditions, although it undergoes dimerization in basic conditions, where its keto form and enolate form react chemically to form a stable dimer [3].  

Being capable of switching between different chemical forms by means of three chemical reactions, a property that is inherent to its molecular structure, the spectrochemistry of firefly oxyluciferin is exceedingly complex. Even when the molecule is in its pure state in model solutions, the emission depends on multiple factors, including pH, polarity and interactions with other molecular species. Being a simple organic molecule with two functional groups, it is surprising that it can exist in as many as six chemical forms that span emission colors from blue (442 nm) to red (626 nm). This tunable emission in model solutions includes the range of emission colors observed with natural bioluminescence (530–640 nm). Additional complication comes from the dependence of the emission on the acidity of the medium; as the pH is changed, the relative ratio of the six chemical species varies, however their emission spectra remain heavily overlapped throughout a broad range of pH. Indeed, the implicit assumption that the emission spectrum of oxyluciferin corresponds to a single species has caused spectral misassignments in the past, and had led to conflicting results.

The disentanglement of the absorption and emission spectra of individual components eventually became possible with the application of a mathematical procedure, multivariate curve resolution, on a great number of absorption and emission spectra of oxyluciferin and its derivatives recorded at varying pH [5,6]. This procedure also required design and development of synthetic routes to prepare chemical analogues of the natural emitter that reflect the effects of various chemical functionalities on the emission properties of individual chemical forms. The model studies indicated that the effects of microenvironment play a major role in determining the emission color of the firefly bioluminescence. Because experimentally it is very difficult to isolate the effects on the emission of a single small molecule from its environment, a special experimental setup (ion storage ring) was used to perform so-called action spectroscopy, which provides information that is unavailable with the solution-state spectroscopy [7]. The result of these experiments provided the electronic spectrum of the emitter in the gas phase, that is, under conditions that closely resemble the state of an isolated emitter molecule sequestered in the active site of the protein. By using this setup, the effects of a single water molecule on the spectrum of oxyluciferin was estimated for the first time. A single water molecule blue-shifts the absorption by approximately 50 nm, which provides an estimate for the effect water molecules could have by going in and out of the active pocket during the bioluminescence reaction [7].

One of the possible sources of additional confusion in the interpretation of the energy profile of the bioluminescence reaction in the past has been the implicit assumption that the reaction pathways in the excited state follow a similar pattern to those in the ground state. A recent study showed, however, that oxyluciferin could become very acidic in the excited state (that is, it behaves as a “super-photoacid”) and undergo tautomerization that is not a viable scenario in the ground state. This excited-state isomerization sheds a new light on the mystery of firefly bioluminescence and it is likely to be the long-sought after key to resolving the keto−enol conundrum of the color-tuning mechanism of firefly bioluminescence [8].

References

[1] Bioluminescence: Chemical Principles and Methods. Shimomura. rev. ed., World Scientific, Singapore, 2012.

[2] Structure and Spectroscopy of Oxyluciferin, the Light Emitter of the Firefly Bioluminescence, Naumov, Panče; Ozawa, Yutaka; Ohkubo, Kei; Fukuzumi, Shunichi, Journal of the American Chemical Society (2009) 131(32), 11590—11605.

[3] Spectra-Structural Effects of the Keto-Enol-Enolate and Phenol-Phenolate Equilibria of Oxyluciferin. Naumov, Panče; Kochunnoonny, Manoj, Journal of the American Chemical Society (2010) 132, 11566–11579.

[4] Why is Firefly Oxyluciferin a Notoriously Labile Substance? Maltsev, Oleg V.; Nath, Naba K.; Naumov, Panče; Hintermann, Lukas, Angewandte Chemie, International Edition (2014) 53, 847—850.

Highlight: Nature Middle East: http://www.nature.com/nmiddleeast/2013/131211/full/nmiddleeast.2013.239....

[5] Deciphering the Protonation and Tautomeric Equilibria of Firefly Oxyluciferin by Molecular Engineering and Multivariate Curve Resolution. Rebarz, Mateusz; Kukovec, Boris-Marko; Maltsev, Oleg V.; Ruckebusch, Cyril, Hintermann, Lukas, Naumov, Pance; Sliwa, Michel, Chemical Science (2013) 4, 3803—3809. (Edge Article, selected as cover article)

[6] Emission Properties of Oxyluciferin and its Derivatives in Water: Revealing the Nature of the Emissive Species in Firefly Bioluminescence. Ghose, Avisek; Rebarz, Mateusz; Maltsev, Oleg V.; Hintermann, Lukas; Ruckebusch, Cyril; Fron, Eduard; Hofkens, Johan; Mély, Yves; Naumov, Panče; Sliwa, Michel; Didier, Pascal, Journal of Physical Chemistry B (2015) 119, 2638—2649. http://pubs.acs.org/doi/abs/10.1021/jp508905m

[7] On the Influence of Water on the Electronic Structure of Firefly Oxyluciferin Anions from Absorption Spectroscopy of Bare and Monohydrated Ions in Vacuo. Støchkel, Kristian; Nygaard Hansen, Christian; Houmøller, Jørgen; Munksgaard Nielsen, Lisbeth; Anggara, Kelvin; Linares, Mathieu; Norman, Patrick; Nogueira, Fernando; Maltsev, Oleg V.; Hintermann, Lukas; Brøndsted Nielsen, Steen; Naumov, Panče; Milne, Bruce F., Journal of the American Chemical Society (2013) 135, 6485—6493.

[8] Photoinduced Dynamics of Oxyluciferin Analogues: Unusual Enol “Super”photoacidity and Evidence for Keto−Enol Isomerization. Solntsev, Kyril M.; Laptenok, Sergey P.; Naumov, Panče, Journal of the American Chemical Society (2012) 134, 16452—16455.