Resonance coherent anti-Stokes Raman scattering spectra of fluorescent biological chromophores: Vibrational evidence for hydrogen bonding of flavin to glucose oxidase and for rapid solvent exchange

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Coherent anti-Stokes Raman scattering (CARS) spectra are reported for flavin adenine dinucleotide (FAD) and glucose oxidase (β-D-glucose:oxygen 1-oxidoreductase; EC 1.1.3.4), in resonance with the 450 nm flavin absorption band. Several isoalloxazine ring modes were observed (1635, 1584, 1507, 1416, and 1359 cm-1). A 12 cm-1 increase in one component of the 1359 cm-1 band upon binding to glucose oxidase was attributed to hydrogen bonding of the N3 proton to a protein acceptor. This interpretation is consistent with deuteration results. Smaller decreases (5-7 cm-1) on binding were observed for the 1635 and 1416 cm-1 modes (and also a 1297 cm-1 mode of deuterated FAD) and were attributed to environmental effects. Deuteration of bound FAD was observed within 5 min of mixing glucose oxidase with D2O, demonstrating ready access to solvent. Lorentzian CARS peaks were observed with ωas at the peak of the 450 nm absorption band, a condition which corresponds to maximum resonance enhancement if the peak corresponds to the envelope of 0-1 vibronic transitions. If ω1 was tuned to the peak, then dispersion lineshapes were observed, reflecting a loss of Raman enhancement relative to the electronic background.

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