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On the influence of water on the electronic structure of firefly oxyluciferin anions from absorption spectroscopy of bare and monohydrated ions in vacuo

Authors: Kristian Støchkel, ChristianHansen, Jørgen Houmøller, Lisbeth Nielsen, Kelvin Anggara, Mathieu Linares, Patrick Norman, Fernando Nogueira, Oleg Maltsev, Lukas Hintermann, Steen Brøndsted Nielsen, Pance Naumov, Bruce F. Milne

Ref.: J. Am. Chem. Soc. 135, 6485-6493 (2013)

Abstract: A complete understanding of the physics underlying the varied colors of firefly bioluminescence remains elusive since it is difficult to disentangle different enzyme–lumophore interactions. Experiments on isolated ions are useful to establish a proper reference when there are no microenvironmental perturbations. Here we use action spectroscopy to compare the absorption by the firefly oxyluciferin lumophore isolated in vacuo and complexed with a single water molecule. Whilst the process relevant to bioluminescence within the luciferase cavity is light emission, the absorption data presented here provide a unique insight into how the electronic states of oxyluciferin are altered by microenvironmental perturbations. For the bare ion we observe broad absorption with a maximum at 548 ± 10 nm, and addition of a water molecule is found to blueshift the absorption by approximately 50 nm (0.23 eV). Test calculations at various levels of theory uniformly predict a blueshift in absorption caused by a single water molecule, but are only qualitatively in agreement with experiment highlighting limitations in what can be expected from methods commonly used in studies on oxyluciferin. Combined molecular dynamics simulations and time–dependent density functional theory calculations closely reproduce the broad experimental peaks and also indicate that the preferred binding site for the water molecule is the phenolate oxygen of the anion. Predicting the effects of microenvironmental interactions on the electronic structure of the oxyluciferin anion with high accuracy is a nontrivial task for theory, and our experimental results therefore serve as important benchmarks for future calculations.

DOI: 10.1021/ja311400t