Hydrogen bonding and spin density distribution in the Q B semiquinone of bacterial reaction centers and comparison with the Q A site

In the photosynthetic reaction center from Rhodobacter sphaeroides, the primary (Q A) and secondary (Q B) electron acceptors are both ubiquinone-10, but with very different properties and functions. To investigate the protein environment that imparts these functional differences, we have applied X-band HYSCORE, a 2D pulsed EPR technique, to characterize the exchangeable protons around the semiquinone (SQ) in the Q A and Q B sites, using samples of 15N-labeled reaction centers, with the native high spin Fe 2+ exchanged for diamagnetic Zn 2+, prepared in 1H 2O and 2H 2O solvent. The powder HYSCORE method is first validated against the orientation-selected Q-band ENDOR study of the Q A SQ by Flores et al. (Biophys. J.2007, 92, 671-682), with good agreement for two exchangeable protons with anisotropic hyperfine tensor components, T, both in the range 4.6-5.4 MHz. HYSCORE was then applied to the Q B SQ where we found proton lines corresponding to T ≈ 5.2, 3.7 MHz and T ≈ 1.9 MHz. Density functional-based quantum mechanics/molecular mechanics (QM/MM) calculations, employing a model of the Q B site, were used to assign the observed couplings to specific hydrogen bonding interactions with the Q B SQ. These calculations allow us to assign the T = 5.2 MHz proton to the His-L190 N ΔH⋯O 4 (carbonyl) hydrogen bonding interaction. The T = 3.7 MHz spectral feature most likely results from hydrogen bonding interactions of O1 (carbonyl) with both Gly-L225 peptide NH and Ser-L223 hydroxyl OH, which possess calculated couplings very close to this value. The smaller 1.9 MHz coupling is assigned to a weakly bound peptide NH proton of Ile-L224. The calculations performed with this structural model of the Q B site show less asymmetric distribution of unpaired spin density over the SQ than seen for the Q A site, consistent with available experimental data for 13C and 17O carbonyl hyperfine couplings. The implications of these interactions for Q B function and comparisons with the Q A site are discussed.(Figure Presented) © 2011 American Chemical Society.