Hydrogen Bonding and Spin Density Distribution in the QB Semiquinone of Bacterial Reaction Centers and Comparison with the QA Site

Abstract

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 Fe²⁺ exchanged for diamagnetic Zn²⁺, prepared in ¹H₂O and ²H₂O 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···O₄ (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.