Probing Protonation Sites of Isolated Flavins Using IR Spectroscopy: From Lumichrome to the Cofactor Flavin Mononucleotide

Authors

  • Dr. Judith Langer,

    1. Technische Universität Berlin, Institut für Optik und Atomare Physik, Hardenbergstraße 36, D-10623 Berlin (Germany)
    2. Current address: Parque Tecnologico de San Sebastian, Paseo Miramon 182, Edif C, 20009 San Sebastian (Spain)
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  • Alan Günther,

    1. Technische Universität Berlin, Institut für Optik und Atomare Physik, Hardenbergstraße 36, D-10623 Berlin (Germany)
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  • Sophie Seidenbecher,

    1. Technische Universität Berlin, Institut für Optik und Atomare Physik, Hardenbergstraße 36, D-10623 Berlin (Germany)
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  • Dr. Giel Berden,

    1. Radboud University Nijmengen, Institute for Molecules and Materials, FELIX Facility, Toernooiveld 7, 6525 ED Nijmegen (The Netherlands)
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  • Prof. Dr. Jos Oomens,

    1. Radboud University Nijmengen, Institute for Molecules and Materials, FELIX Facility, Toernooiveld 7, 6525 ED Nijmegen (The Netherlands)
    2. University of Amsterdam, Science Park 904, 1098 XH Amsterdam (The Netherlands)
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  • Prof. Dr. Otto Dopfer

    Corresponding author
    1. Technische Universität Berlin, Institut für Optik und Atomare Physik, Hardenbergstraße 36, D-10623 Berlin (Germany)
    • Technische Universität Berlin, Institut für Optik und Atomare Physik, Hardenbergstraße 36, D-10623 Berlin (Germany)

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Abstract

Infrared spectra of the isolated protonated flavin molecules lumichrome, lumiflavin, riboflavin (vitamin B2), and the biologically important cofactor flavin mononucleotide are measured in the fingerprint region (600–1850 cm−1) by means of IR multiple-photon dissociation (IRMPD) spectroscopy. Using density functional theory calculations, the geometries, relative energies, and linear IR absorption spectra of several low-energy isomers are calculated. Comparison of the calculated IR spectra with the measured IRMPD spectra reveals that the N10 substituent on the isoalloxazine ring influences the protonation site of the flavin. Lumichrome, with a hydrogen substituent, is only stable as the N1-protonated tautomer and protonates at N5 of the pyrazine ring. The presence of the ribityl unit in riboflavin leads to protonation at N1 of the pyrimidinedione moiety, and methyl substitution in lumiflavin stabilizes the tautomer that is protonated at O2. In contrast, flavin mononucleotide exists as both the O2- and N1-protonated tautomers. The frequencies and relative intensities of the two C[DOUBLE BOND]O stretch vibrations in protonated flavins serve as reliable indicators for their protonation site.

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