Nuclear hyperfine and quadrupole tensor characterization of the nitrogen hydrogen bond donors to the semiquinone of the Q_B site in bacterial reaction centers: A combined X- and S-band ^14,15N ESEEM and DFT study

The secondary quinone anion radical Q_B^- (SQ _B) in reaction centers of Rhodobacter sphaeroides interacts with N_δ of His-L190 and N_p (peptide nitrogen) of Gly-L225 involved in hydrogen bonds to the Q_B carbonyls. In this work, S-band (∼3.6 GHz) ESEEM was used with the aim of obtaining a complete characterization of the nuclear quadrupole interaction (nqi) tensors for both nitrogens by approaching the cancelation condition between the isotropic hyperfine coupling and ¹⁴N Zeeman frequency at lower microwave frequencies than traditional X-band (9.5 GHz). By performing measurements at S-band, we found a dominating contribution of N_δ in the form of a zero-field nqi triplet at 0.55, 0.92, and 1.47 MHz, defining the quadrupole coupling constant K = e²qQ/4h = 0.4 MHz and associated asymmetry parameter η = 0.69. Estimates of the hyperfine interaction (hfi) tensors for N_δ and N_p were obtained from simulations of 1D and 2D ^14,15N X-band and three-pulse ¹⁴N S-band spectra with all nuclear tensors defined in the SQ_B g-tensor coordinate system. From simulations, we conclude that the contribution of N_p to the S-band spectrum is suppressed by its strong nqi and weak isotropic hfi comparable to the level of hyperfine anisotropy, despite the near-cancelation condition for N_p at S-band. The excellent agreement between our EPR simulations and DFT calculations of the nitrogen hfi and nqi tensors to SQ _B is promising for the future application of powder ESEEM to full tensor characterizations.