Preparation and spectroscopic studies of 5-deazaflavin isotopologues in isotropic and anisotropic surroundings

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Deazaflavins are important analogues of the naturally occurring flavins: riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The use of 5-deazaflavins as replacement coenzymes in a number of flavoproteins has proven particularly valuable in unraveling and manipulating their reaction mechanisms. It was frequently observed that one-electron-transfer reactions in flavoproteins are impeded with 5-deazaflavin as 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 needs to be reevaluated given our recent success in (indirectly) observing a one-electron reduced (paramagnetic) species using photo-chemically induced dynamic nuclear polarization (photo-CIDNP) NMR. We propose characterizing the electronic structure of this radical by using electron paramagnetic resonance (EPR) derived hyperfine spectroscopies as well as photo-CIDNP NMR. A library of selectively stable-isotope (13C, 15N) labeled 5-deazaflavins required for this purpose will be synthesized and incorporated into various blue-light photoreceptor domains (LOV and BLUF) that are of interest in optogenetic applications. Using two-dimensional NMR, we plan to unravel structural traits of photo-adduct formation in wild-type and mutant LOV domains harboring 5-deazaFMN as chromophore.