Determining large hyperfine interactions of a model flavoprotein in the semiquinone state using pulse EPR (electron...

Flavoproteins are a versatile class of proteins involved in numerous biological processes, including redox reactions, electron transfer, and signal transduction, often relying on their ability to stabilize different oxidation states of their flavin cofactor. A critical feature of flavin cofactors is their capacity to achieve, within particular protein environments, a semiquinone state that plays a pivotal role in mediating single-electron transfer events and that is key to understanding flavoprotein reactivity. Hyperfine interactions between the unpaired electron and magnetic nuclei in the isoalloxazine ring provide valuable insights into the semiquinone state and its mechanistic roles. This study investigates the hyperfine interactions of isotopically labeled flavodoxin (Fld) with ${}^{13}C$ and ${}^{15}N$ in specific positions of the flavin mononucleotide (FMN) ring using advanced electron paramagnetic resonance (EPR) techniques. The combination of continuous-wave (CW) EPR at the X-band and ELDOR-detected NMR and HYSCORE at the Q-band revealed a strong and anisotropic hyperfine interaction with the nucleus of ${}^{13}C$ at 4a and yielded principal tensor values of 40, $-13.5$ , and $-9$ MHz, the first of which is associated with the axis perpendicular to the flavin plane. On the other hand, as predicted, the hyperfine interaction with the ${}^{13}C$ nucleus in position 2 was minimal. Additionally, HYSCORE experiments on ${}^{15}N$ -FMN-labeled Fld provided precise axial hyperfine parameters, i.e., (74, 5.6, 5.6) $\mathrm{MHz}$ for ${}^{15}N$ (5) and (38, 3.2, 3.2) $\mathrm{MHz}$ for ${}^{15}N$ (10). These were used to refine quadrupole tensor values for ${}^{14}N$ nuclei through isotope-dependent scaling. These results showcase the potential of combining CW EPR, ELDOR-detected NMR, and HYSCORE with isotopic labeling to probe electronic and nuclear interactions in flavoproteins. The new data complete and refine the existing experimental map for the electronic structure of the flavin cofactor and expose systematic divergences between the calculated and experimental values of hyperfine couplings of the atoms that contribute most to the semi-occupied orbital (SOMO). This could indicate a slight but significant shift in the unpaired electron density from position 4a towards the central nitrogens of the pyrazine ring as compared with the calculations. These results highlight the importance of integrating computational and experimental approaches to refine our understanding of flavin cofactor reactivity.