Excited State Vibrations of Isotopically Labeled FMN Free and Bound to a Light–Oxygen–Voltage (LOV) Protein

James N. Iuliano, Christopher Hall, Dale Green, Garth Jones, Andras Lukacs, Boris Illarionov, Adelbert Bacher, M Fischer, Jarrod B. French, Peter J. Tonge, Steve Meech

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Abstract

Flavoproteins are important blue light sensors in photobiology and play a key role in optogenetics. The characterization of their excited state structure and dynamics is thus an important objective. Here, we present a detailed study of excited state vibrational spectra of flavin mononucleotide (FMN), in solution and bound to the LOV-2 (Light-Oxygen-Voltage) domain of Avena sativa phototropin. Vibrational frequencies are determined for the optically excited singlet state and the reactive triplet state, through resonant ultrafast femtosecond stimulated Raman spectroscopy (FSRS). To assign the observed spectra, vibrational frequencies of the excited states are calculated using density functional theory, and both measurement and theory are applied to four different isotopologues of FMN. Excited state mode assignments are refined in both states, and their sensitivity to deuteration and protein environment are investigated. We show that resonant FSRS provides a useful tool for characterizing photoactive flavoproteins and is able to highlight chromophore localized modes and to record hydrogen/deuterium exchange.
Original languageEnglish
Pages (from-to)7152–7165
Number of pages14
JournalJournal of Physical Chemistry B
Volume124
Issue number33
Early online date27 Jul 2020
DOIs
Publication statusPublished - 20 Aug 2020

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