Methyl groups matter: Photo-CIDNP characterizations of the semiquinone radicals of FMN and demethylated FMN analogs

Abstract

In this contribution, the relative hyperfine couplings are determined for the 1H nuclei of the flavin mononucleotide (FMN) radical in an aqueous environment. In addition, three structural analogs with different methylation patterns are characterized and the influence of the substituents at the isoalloxazine moiety on the electronic structure of the radicals is explored. By exploiting nuclear hyperpolarization generated via the photo-CIDNP (chemically induced dynamic nuclear polarization) effect, it is possible to study the short-lived radical species generated by in situ light excitation. Experimental data are extracted by least-squares fitting and supported by quantum chemical calculations and published values from electron paramagnetic resonance and electron-nuclear double resonance. Furthermore, mechanistic details of the photoreaction of the investigated flavin analogs with l-tryptophan are derived from the photo-CIDNP spectra recorded at different pH values. Thereby, the neutral and anionic radicals of FMN and three structural analogs are, for the first time, characterized in terms of their electronic structure in an aqueous environment.

Coupled Methyl Group Rotation in FMN Radicals Revealed by Selective Deuterium Labeling

“…In the spectrum recorded at g z , however, only one signal close to the free deuteron Larmor frequency is observed, which exhibits a total width of 0.5 MHz and has a shoulder at 0.25 MHz. Although DFT calculations suggest and photo-CIDNP studies confirm that the hf tensor of 2 H­(9) is generally larger than that from the three 2 H­(7α) nuclei, an unequivocal assignment of the individual hf principal values is not possible from these two spectra alone.…”

“…Hence, we reinvestigated the LOV* protein with ENDOR spectroscopy at low temperatures. The electron spin density situated on the three 7α methyl-group protons is supposed to be small as measurements of the isotropic hyperfine coupling using photo-chemically induced dynamic nuclear polarization (photo-CIDNP) imply . Therefore, their ENDOR resonances are expected to overlap with those of other nuclei with small proton hfcs in the matrix ENDOR line.…”

Understanding flavin electronic structure and spectra

“…In‐line with recent advances, the use of a segmented contracted basis set, also known as a Jensen basis set, 178 could be a better alternative for calculation of NMR properties for flavin systems 179 . Last but not the least, GIAO methods were shown to replicate hyperfine coupling constants for flavin semiquinone states in isolated systems, 85 COSMO solvent models 180 or a protein‐embedded system 181 …”

Non-classical disproportionation revealed by photo-chemically induced dynamic nuclear polarization NMR

“…NMR and photo-chemically induced dynamic nuclear polarization (photo-CIDNP) experiments were performed as described previously (Pompe et al, 2019), using a Bruker Avance III HD 600 MHz NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) operating at 14.1 T. Light excitation was achieved by coupling the output of a nanosecond-pulsed laser system, comprising an Nd:YAG laser source (Surelite I, Continuum, Santa Clara, CA, USA) in combination with a broadband optical parametric oscillator (OPO) (Continuum OPO PLUS), into an optical fiber with a diameter of 1 mm (Thorlabs, Dachau, Germany). The optical fiber was inserted into the NMR tube via a coaxial insert (Wilmad WGS-5BL).…”

“…2 affords riboflavin by a mechanistically unique dismutation that is catalyzed by the enzyme riboflavin synthase and does not require any cosubstrates or cofactors (Plaut and Harvey, 1971). Even more surprisingly, the dismutation affording 4 from 2 can also proceed non-enzymatically in neutral or acidic aqueous solution under an inert gas atmosphere (Rowan and Wood, 1963;Kis et al, 2001). 6,7-Dimethyl-8-ribityllumazine (2) acts as cofactor of lumazine protein (LumP), an optical transponder involved in bioluminescence of certain marine bacteria (Koka and Lee, 1979;Small et al, 1980).…”