Abstract
Photosynthetic reaction centers from Rhodobacter sphaeroides have identical ubiquinone-10 molecules functioning as primary (Q(A)) and secondary (Q(B)) electron acceptors. X-band 2D pulsed EPR spectroscopy, called HYSCORE, was applied to study the interaction of the Q(B) site semiquinone with nitrogens from the local protein environment in natural and (15)N uniformly labeled reactions centers. (14)N and (15)N HYSCORE spectra of the Q(B) semiquinone show the interaction with two nitrogens carrying transferred unpaired spin density. Quadrupole coupling constants estimated from (14)N HYSCORE spectra indicate them to be a protonated nitrogen of an imidazole residue and amide nitrogen of a peptide group. (15)N HYSCORE spectra allowed estimation of the isotropic and anisotropic couplings with these nitrogens. From these data, we calculated the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and analyzed the contribution of different factors to the anisotropic hyperfine tensors. The hyperfine coupling of other protein nitrogens with the semiquinone is weak (<0.1 MHz). These results clearly indicate that the Q(B) semiquinone forms hydrogen bonds with two nitrogens and provide quantitative characteristics of the hyperfine couplings with these nitrogens, which can be used in theoretical modeling of the Q(B) site. On the basis of the quadrupole coupling constant, one nitrogen can only be assigned to N(delta) of His-L190, consistent with all existing structures. However, we cannot specify between two candidates the residue corresponding to the second nitrogen. Further work employing multifrequency spectroscopic approaches or selective isotope labeling would be desirable for unambiguous assignment of this nitrogen.