Determining large hyperfine interactions of a model flavoprotein in the semiquinone state by pulse-EPR techniques 

Abstract. 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 is key to understanding flavoprotein reactivity.

Hyperfine interactions between the unpaired electron in the semiquinone state and magnetic nuclei in the isoalloxazine ring provide valuable insights into the electronic structure of this intermediate and its mechanistic roles. This study investigates the hyperfine interactions of isotopically labeled flavodoxin (Fld) with ¹³C and ¹⁵N at specific positions of the flavin mononucleotide (FMN) ring using advanced electron paramagnetic resonance (EPR) techniques. The combination of Continuous wave (CW) EPR at X-band and ELDOR-detected NMR and HYSCORE at Q-band revealed a strong and anisotropic hyperfine interaction with the nucleus ¹³C at 4a and yielded principal tensor values of 40, -13.5 and -9 MHz, the first of which is associated to the axis perpendicular to the flavin plane. On the other hand, as predicted, the hyperfine interaction with the ¹³C nucleus at position 2 was minimal. Additionally, HYSCORE experiments on ¹⁵N-FMN labeled Fld provided precisely axial hyperfine parameters, (74, 5.6, 5.6) MHz for ¹⁵N(5) and (38, 3.2, 3.2) MHz for ¹⁵N(10). These were used to refine quadrupole tensor values for ¹⁴N nuclei via isotope-dependent scaling. These results showcase the potential of combining CW-EPR, ELDOR-detected NMR, and HYSCORE with isotopic labelling 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 most contributing to the SOMO. This could indicate a slight but significant shift of the unpaired electron density from position 4a towards the central nitrogens of the pyrazine ring as compared to the calculations. These results highlight the importance of integrating computational and experimental approaches to refine our understanding of flavin cofactor reactivity.