Expanding Reaction Horizons: Evidence of the 5-Deazaflavin Radical Through Photochemically Induced Dynamic Nuclear Polarization

Deazaflavins are important analogues of the naturally occurring flavins: riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The use of 5-deazaflavin as a replacement coenzyme in a number of flavoproteins has proven particularly valuable in unraveling and manipulating their reaction mechanisms. It was frequently reported that one-electron-transfer reactions in flavoproteins are impeded with 5-deazaflavin as the cofactor. Based on these findings, it was concluded that the 5-deazaflavin radical is significantly less stable compared to the respective flavin semiquinone and quickly re-oxidizes or undergoes disproportionation. The long-standing paradigm of 5-deazaflavin being solely a two-electron/hydride acceptor/donor-"a nicotinamide in flavin clothing"-needs to be re-evaluated now with the indirect observation of a one-electron-reduced (paramagnetic) species using photochemically induced dynamic nuclear polarization (photo-CIDNP) 1H nuclear magnetic resonance (NMR) under biologically relevant conditions. Using photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR spectroscopy, we found unambiguous evidence of a one-electron-reduced 5-deazaFMN species. Time-resolved experiments provide a hyperfine mapping for the protons in the aromatic moiety; the relative isotropic hyperfine couplings are correlated to data from density functional theory.+image