Photochemistry and Photobiology, 2013, 89: 319–325 Are the Bio- and Chemiluminescence States of the Firefly Oxyluciferin the Same as the Fluorescence State? Isabelle Navizet* 1,2 , Daniel Roca-Sanjuán 3 , Ling Yue 4 , Ya-Jun Liu 4 , Nicolas Ferré 5 and Roland Lindh 3 1 Molecular Science Institute School of Chemistry, University of the Witwatersrand, Johannesburg, South Africa 2 Université Paris-Est, Laboratoire Modélisation et Simulation Multi Echelle, Marne-la-Vallée, France 3 Department of Chemistry—Ångström, Theoretical Chemistry Programme, Uppsala University, Uppsala, Sweden 4 Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, China 5 Aix-Marseille Université, Institut de Chimie Radicalaire Campus Saint-Jérome Case 521, Marseille Cedex 20, France Received 21 June 2012, accepted 5 October 2012, DOI: 10.1111/php.12007 ABSTRACT A usual strategy in both experimental and theoretical studies on bio- and chemiluminescence is to analyze the fluorescent properties of the bio- and chemiluminescence reaction prod- uct. Recent findings in a coelenteramide and Cypridina oxyluciferin model raise a concern on the validity of this procedure, showing that the light emitters in each of these luminescent processes might differ. Here, the thermal decom- position path of the firefly dioxetanone and the light emission states of the Firefly oxyluciferin responsible for the bio-, chemiluminescence, and fluorescence of the molecule are characterized using ab initio quantum chemistry and hybrid quantum chemistry/molecular mechanics methods to deter- mine if the scenario found in the coelenteramide and Cypri- dina oxyluciferin study does also apply to the Firefly bioluminescent systems. The results point out to a unique emission state in the bio-, chemiluminescence, and fluores- cence phenomena of the Firefly oxyluciferin and, therefore, using fluorescence properties of this system is reasonable. INTRODUCTION Firefly bioluminescence is a phenomenon resulting from an enzyme-catalyzed chemical reaction leading to a molecule in its excited state, the Firefly oxyluciferin (Fl-oxy*) (see Fig. 1). Increasing interest on the Firefly bioluminescence in particular and bioluminescence occurring in living organisms in general is sum- marized by the publication of not less than five reviews in the last year (1–5). To our knowledge, all theoretical studies on the light emission from oxyluciferin in vacuo, solvent, or inside the protein use the oxyluciferin ground state (S 0 ) equilibrium structure as start- ing geometry for the optimization of the first excited state (S 1 ). This structure is found to be planar around the C 4 atom (see Fig. 1 for numbering) with a characteristic C 4 –O 11 bond close to a double bond and its electronic structure is characterized by a p?p* excitation among orbitals from the rings. Because of the difficulty to acquire the fluorescence spectrum of the real emitter, many experi- ments on bio- or chemiluminescence also proceed by analyzing the fluorescent properties of the oxyluciferin molecule, which is the decomposition product (see Refs. (2) and (6) and references therein). Recently, we have shown that for a small model of coelen- teramide and Cypridina luciferin, the chemiluminescent and fluo- rescent structures are not the same (6). As defined in that contribution, the “fluorescent state” corresponds to the relaxed singlet excited state obtained from the light absorption of the product of the reaction and the “chemiluminescent state” is the equilibrium structure of the singlet S 1 state of the oxy-compound reached after the cleavage of the carbon dioxide from the dioxe- tanone (DO) compound. For the small model of the coelentera- mide and Cypridina luciferin, the chemiluminescent species, is still bent and the C–O bond is longer than a double bond, whereas the fluorescent state is planar and has double bond char- acter (Fig. 2). In addition, the chemiluminescent state features a charge transfer (CT) to the CO group in contrast to the fluores- cent state. Do these results apply to the Firefly luciferin? Studies concerned on the mechanism of the bio- and chemilu- minescent reactions have been mainly performed on model com- pounds (7–13). A few attempts have been carried out to unveil the mechanism of the Firefly dioxetanone (Fl-DO) decomposi- tion leading to the Firefly oxyluciferin (Fl-oxy). The first compu- tations on the transition state (TS) of the Fl-DO decomposition were carried out at the density functional theory (DFT) B3LYP/6 –31+G(d) level of theory, with a reported barrier of 5.1 kcal mol À1 (12). The authors also performed a fine analysis of the thermal decomposition of the p- and m-phenolate dioxeta- none. In 2008, a communication on calculations at high level of theory, complete active space self-consistent field and a complete active space second-order perturbation theory (CASSCF/CASPT2) with an active space comprising 12 electrons in 12 p orbitals (hereafter, 12in12), focused on the first part of the reaction and characterized the structures of the ground state (GS) Fl-DO, the TS, and the minimum energy conical intersection (MECI) on the pathway to reach the first singlet excited state of Fl-oxy (Fl-oxy*) (14). In 2011, Min et al. reported a DFT M06 study on the formation and the decomposition reactions of the Fl-DO in the gas phase and in a solvent model. The authors proposed two dif- ferent paths for the decomposition reaction, but focusing on the *Corresponding author email: isabelle.navizet@wits.ac.za (Isabelle Navizet) © 2012 Wiley Periodicals, Inc. 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