Contributions of the 8-Methyl Group to the Vibrational Normal Modes of Flavin Mononucleotide and Its 5-Methyl Semiquinone Radical

Abstract

Resonance Raman spectroscopy is a powerful tool to investigate flavins and flavoproteins, and a good understanding of the flavin vibrational normal modes is essential for the interpretation of the Raman spectra. Isotopic labeling is the most effective tool for the assignment of vibrational normal modes, but such studies have been limited to labeling of rings II and III of the flavin isoalloxazine ring. In this paper, we report the resonance and pre-resonance Raman spectra of flavin mononucleotide (FMN) and its N5-methyl neutral radical semiquinone (5-CH 3FMN(*)), of which the 8-methyl group of ring I has been deuterated. The experiments indicate that the Raman bands in the low-frequency region are the most sensitive to 8-methyl deuteration. Density functional theory (DFT) calculations have been performed on lumiflavin to predict the isotope shifts, which are used to assign the calculated normal modes to the Raman bands of FMN. A first assignment of the low-frequency Raman bands on the basis of isotope shifts is proposed. Partial deuteration of the 8-methyl group reveals that the changes in the Raman spectra do not always occur gradually. These observations are reproduced by the DFT calculations, which provide detailed insight into the underlying modifications of the normal modes that are responsible for the changes in the Raman spectra. Two types of isotopic shift patterns are observed: either the frequency of the normal mode but not its composition changes or the composition of the normal mode changes, which then appears at a new frequency. The DFT calculations also reveal that the effect of H/D-exchange in the 8-methyl group on the composition of the vibrational normal modes is affected by the position of the exchanged hydrogen, i.e., whether it is in or out of the isoalloxazine plane.

Deep ultraviolet initiated excited state dynamics of riboflavin and flavin mononucleotide

“…Weigel et al have done the assignment of RR bands of RF and FAD using the same B3LYP/TZVP/PCM model with the addition of four water molecules around the electronegative atoms of isoalloxazine ring. Our assignments of UVRR bands of RF are consistent with their assignment and also with other calculations . The vibrations which have less than 10% contribution have not been included in the assignment table.…”

“…A new band appears in the form of a shoulder at 1,281 cm −1 with 270.4 nm (Figure S2) and other higher excitation wavelengths. The Raman band at 1,281 cm −1 for flavins was previously reported with UV and visible excitations . This Raman band is resonantly enhanced by low energy electronic transition centered around 280 nm within the examining absorption band and are not considered further.…”

Methyl rotors in flavoproteins

“…Evidence for methyl rotation in flavins and related systems is scarce. The relationship of the presence of a methyl substituent at position 8 in flavin rings with different physicochemical properties of the molecule (electronic structure, dipolar moment, vibrational modes) was characterized by fluorescence measurements, Stark spectroscopy, resonance Raman spectroscopy and DFT calculations, [53][54][55][56][57] and its functional consequences described. 58,59 Nevertheless, these techniques are not able to detect the effects of methyl dynamics as this ENDOR study does.…”

Enhanced photocatalytic activity of a self-stabilized synthetic flavin anchored on a TiO2 surface

“…In a typical experiment, 20 mg of the oxide catalyst were spread uniformly over a 0.79 cm 2 area of a custom-built photocatalyst holder, placed at the bottom of the reactor (Fig. [18][19][20] This statement is bolstered by comparing the spectrum with that of a similar synthetic flavin, (denoted here as MSH-197, see Fig. A 2 mL aliquot of ethanol was injected into the reactor by a microsyringe through a septum.…”