Structure and spectroscopy of oxyluciferin, the light emitter of the firefly bioluminescence

Pance Naumov, Yutaka Ozawa, Kei Ohkubo, Shunichi Fukuzumi

    Research output: Contribution to journalArticle

    Abstract

    The crystal structures of the pure, unsubstituted firefly emitter oxyluciferin (OxyLH2) and its 5-methyl analogue (MOxyLH2) were determined for the first time to reveal that both molecules exist as pure trans-enol forms, enol-OxyLH2 and enol-MOxyLH2, assembled as head-to-tail hydrogen-bonded dimers. Their steady-state absorption and emission spectra (in solution and in the solid state) and nanosecond time-resolved fluorescence decays (in solution) were recorded and assigned to the six possible trans chemical forms of the emitter and its anions. The spectra of the pure emitter were compared to its bioluminescence and fluorescence spectra when it is complexed with luciferase from the Japanese firefly (Luciola cruciata) and interpreted in terms of the intermolecular interactions based on the structure of the emitter in the luciferase active site. The wavelengths of the emission spectral maxima of the six chemical forms of OxyLH2 are generally in good agreement with the theoretically predicted energies of the S0-S1 transitions and range from the blue to the red regions, while the respective absorption maxima range from the ultraviolet to the green regions. It was confirmed that both neutral forms, phenol-enol and phenol-keto, are blue emitters, whereas the phenolate-enol form is yellow-green emitter. The phenol-enolate form, which probably only exists as a mixture with other species, and the phenolate-enolate dianion are yellow or orange emitters with close position of their emission bands. The phenolate-keto form always emits in the red region. The concentration ratio of the different chemical species in solutions of OxyLH2 is determined by several factors which affect the intricate triple chemical equilibrium, most notably the pH, solvent polarity, hydrogen bonding, presence of additional ions, and π-π stacking. Due to the stabilization of the enol group of the 4-hydroxythiazole ring by hydrogen bonding to the proximate adenosine monophosphate, which according to the density functional calculations is similar to that due to the dimerization of two enol molecules observed in the crystal, the phenolate ion of the enol tautomer, which is the predominant ground-state species within the narrow pH interval 7.44-8.14 in buffered aqueous solutions, is the most probable emitter of the yellow-green bioluminescence common for most wild-type luciferases. This conclusion is supported by the bioluminescence/fluorescence spectra and the NMR data, as well the crystal structures of OxyLH2 and MOxyLH 2, where the conjugated acid (phenol) of the emitter exists as pure enol tautomer.

    Original languageEnglish (US)
    Pages (from-to)11590-11605
    Number of pages16
    JournalJournal of the American Chemical Society
    Volume131
    Issue number32
    DOIs
    StatePublished - Aug 19 2009

    Fingerprint

    Fireflies
    Bioluminescence
    Phenol
    Phenols
    Spectrum Analysis
    Luciferases
    Spectroscopy
    Light
    Fluorescence
    Hydrogen Bonding
    Hydrogen bonds
    Crystal structure
    Ions
    Firefly Luciferases
    Rubiaceae
    Molecules
    Dimerization
    Adenosine Monophosphate
    Dimers
    Ground state

    ASJC Scopus subject areas

    • Catalysis
    • Chemistry(all)
    • Biochemistry
    • Colloid and Surface Chemistry

    Cite this

    Structure and spectroscopy of oxyluciferin, the light emitter of the firefly bioluminescence. / Naumov, Pance; Ozawa, Yutaka; Ohkubo, Kei; Fukuzumi, Shunichi.

    In: Journal of the American Chemical Society, Vol. 131, No. 32, 19.08.2009, p. 11590-11605.

    Research output: Contribution to journalArticle

    Naumov, Pance ; Ozawa, Yutaka ; Ohkubo, Kei ; Fukuzumi, Shunichi. / Structure and spectroscopy of oxyluciferin, the light emitter of the firefly bioluminescence. In: Journal of the American Chemical Society. 2009 ; Vol. 131, No. 32. pp. 11590-11605.
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