Published: February 25, 2011 r2011 American Chemical Society 2155 dx.doi.org/10.1021/jp110120c | J. Phys. Chem. A 2011, 115, 2155–2159 ARTICLE pubs.acs.org/JPCA Absorption Spectrum of the Firefly Luciferin Anion Isolated in Vacuo Kristian Støchkel, †, * Bruce F. Milne, ‡, * and Steen Brøndsted Nielsen † † Department of Physics and Astronomy, Aarhus University, Ny Munkegade, DK-8000 Aarhus C, Denmark ‡ Centre for Computational Physics, Physics Department, University of Coimbra, Rua Larga, 3004-516, Coimbra, Portugal b S Supporting Information ’ INTRODUCTION Luciferin (LH 2 , Figure 1) is the substrate molecule for the enzymatic reaction responsible for the characteristic yellow light emission from fireflies. 1 In aqueous solution the carboxylic acid moiety of the molecule is deprotonated at physiological pH, with the anion carrying a unit negative charge. LH 2 undergoes a Mg 2þ - mediated reaction with adenosine triphosphate in the active site of the luciferase enzyme, releasing inorganic pyrophosphate. The resulting luciferyl adenylate compound reacts with molecular oxygen, liberating one molecule each of adenosine monophosphate and carbon dioxide. This final step yields the product molecule, oxyluciferin, in a singlet excited state, and it is this excited product molecule that emits a photon of light as it relaxes to the ground state and produces the firefly’s bioluminescence. Both luciferin and oxyluciferin are expected to display charge- transfer (CT) character in the excited state where charge density has been moved between the five-membered thiazolyl ring and the fused benzothiazolyl ring system. Oxyluciferin is extremely unstable and has only recently been synthesized, isolated and studied spectroscopically by Naumov et al. 2 In their study, the absorption and fluorescence spectra of oxyluciferin were obtained in a range of solvents. An emission wavelength of 553 nm was obtained for aqueous solution, in good agreement with the Photinus pyralis bioluminescent maximum of 562 nm, while in organic (less-polar) solvents this value was reduced by more than 80 nm. Even though different species of bioluminescent beetles all use the same luciferin, the emission spectrum can vary greatly, e.g., the light emitted from the firefly Photuris pennsylvanica is green (538 nm) while that emitted by the railroad worm Phrixotrix hirtus is red (623 nm). 3 Probably the most studied species is the North American firefly P. pyralis which produces a yellow-green light having a wavelength of 562 nm. 3 This variability is most likely linked to alterations in the tertiary structure of the luciferase enzyme arising from interspecies differences in the corresponding amino acid sequence, 4 although a number of other factors have been found to play a role in determining the emission color for the luciferase/luciferin reaction including temperature, pH and the presence of divalent metal ions such as Cu 2þ . 4-6 To better understand the bioluminescence phenomenon and the influence of a chemical environment, detailed theoretical work has been carried out on both luciferin and oxyluciferin, isolated in vacuum or in solvents 7-12 or, more recently, em- bedded in the protein environment. 13-16 However, to date there have been no experimental gas-phase data to compare with and thus no reference spectra free of microenvironmental effects with which to evaluate the theoretical methods used. Especially, information on the intrinsic electronic properties of the ligand molecules is relevant as the importance of nearby molecules or charges can then be directly inferred. Received: October 22, 2010 Revised: January 25, 2011 ABSTRACT: The excited-state physics of the firefly luciferin anion depends on its chemical environment, and it is therefore important to establish the intrinsic behavior of the bare ion. Here we report electronic absorption spectra of the anion isolated in vacuo obtained at an electrostatic ion storage ring and an accelerator mass spectrometer where ionic dissociation is monitored on a long time scale (from 33 μs and up to 3 ms) and on a short time scale (0-3 μs), respectively. In the ring experiment the yield of all neutrals (mainly CO 2 ) as a function of wavelength was measured whereas in the single pass experiment, the abundance of daughter ions formed after loss of CO 2 was recorded to provide action spectra. We find maxima at 535 and 265 nm, and that the band shape is largely determined by the sampling time interval, which is due to the kinetics of the dissociation process. Calculations at the TD-B3LYP/TZVPPþþ level predict maximum absorption at 533 and 275 nm for the carboxylate isomer in excellent agreement with the experimental findings. The phenolate isomer lies higher in energy by 0.22 eV, and also its absorption maximum is calculated to be at 463 nm, which is far away from the experimental value. Our data serve to benchmark future theoretical models for bioluminescence from fireflies.