Hyperfine and nuclear quadrupole tensors of nitrogen donors in the Q _A site of bacterial reaction centers: Correlation of the histidine N_δ tensors with hydrogen bond strength

X- and Q-band pulsed EPR spectroscopy was applied to study the interaction of the Q_A site semiquinone (SQ_A) with nitrogens from the local protein environment in natural abundance ¹⁴N and in ¹⁵N uniformly labeled photosynthetic reaction centers of Rhodobacter sphaeroides. The hyperfine and nuclear quadrupole tensors for His-M219 N _δ and Ala-M260 peptide nitrogen (N_p) were estimated through simultaneous simulation of the Q-band ¹⁵N Davies ENDOR, X- and Q-band ^14,15N HYSCORE, and X-band ¹⁴N three-pulse ESEEM spectra, with support from DFT calculations. The hyperfine coupling constants were found to be a(¹⁴N) = 2.3 MHz, T = 0.3 MHz for His-M219 N_δ and a(¹⁴N) = 2.6 MHz, T = 0.3 MHz for Ala-M260 N_p. Despite that His-M219 N_δ is established as the stronger of the two H-bond donors, Ala-M260 N_p is found to have the larger value of a(¹⁴N). The nuclear quadrupole coupling constants were estimated as e²Qq/4h = 0.38 MHz, η = 0.97 and e ²Qq/4h = 0.74 MHz, η = 0.59 for His-M219 N_δ and Ala-M260 N_p, respectively. An analysis of the available data on nuclear quadrupole tensors for imidazole nitrogens found in semiquinone-binding proteins and copper complexes reveals these systems share similar electron occupancies of the protonated nitrogen orbitals. By applying the Townes-Dailey model, developed previously for copper complexes, to the semiquinones, we find the asymmetry parameter η to be a sensitive probe of the histidine N _δ-semiquinone hydrogen bond strength. This is supported by a strong correlation observed between η and the isotropic coupling constant a(¹⁴N) and is consistent with previous computational works and our own semiquinone-histidine model calculations. The empirical relationship presented here for a(¹⁴N) and η will provide an important structural characterization tool in future studies of semiquinone-binding proteins.