Hydrogen bonds between nitrogen donors and the semiquinone in the Q _i-site of the bc₁ complex

The ubisemiquinone stabilized at the Q_i-site of the bc ₁ complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of ¹⁴N and ¹⁵N have been studied by X-band (∼9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the ¹⁴N involved in H-bond formation and to assign it unambiguously to the N^ε of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and N^δ in other quinone-processing sites. The experiments with ¹⁵N showed that the N^ε of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the N^ε of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38 ± 0.13 Å. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone.A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.