Hydrogen Bonding and Spin Density Distribution in the Q_BSemiquinone of Bacterial Reaction Centers and Comparison with the Q_ASite

Abstract

In the photosynthetic reaction center from Rhodobacter sphaeroides, the primary (QA) and secondary (QB) 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 QA and QB 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 QA 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 QB 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 QB site, were used to assign the observed couplings to specific hydrogen bonding interactions with the QB SQ. These calculations allow us to assign the T=5.2 MHz proton to the His-L190 NδH…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 QB site show less asymmetric distribution of unpaired spin density over the SQ than seen for the QA site, consistent with available experimental data for 13C and 17O carbonyl hyperfine couplings. The implications of these interactions for QB function and comparisons with the QA site are discussed.

Hydrogen Bonding between the QBSite Ubisemiquinone and Ser-L223 in the Bacterial Reaction Center: A Combined Spectroscopic and Computational Perspective

“…Due to its rotational freedom the Ser-L223 hydroxyl group can be rotated away from hydrogen bond donation to O 1 and, indeed, in some models it has been proposed to hydrogen bond to Asp-L213 instead. (11, 12) Based on the previous report of a hydrogen bond to Ser-L223 by Q-band ENDOR (13) we assumed in a previous publication (18) that the hydroxyl group of this residue is rotated such that a hydrogen bond donation occurs to the SQ B O 1 atom. This hydrogen-bonded model is denoted WT-HB.…”

“…(18) The instrumentation, pulse sequences and spectral processing for X-band 1D 4-pulse ESEEM (π/2-τ-π/2-t-π-t-π/2-echo) and 2D 4-pulse ESEEM (HYSCORE) (π/2-τ-π/2-t 1 -π-t 2 -π/2-echo) were also as described. (18) Pulsed ENDOR spectra of the semiquinones were obtained using Davies (π- t -π/2-τ-π-τ-echo) and Mims (π/2-τ-π/2- t -π/2-τ) sequences with different pulse lengths. In addition, a radio frequency π pulse is applied during the time interval t in both sequences.…”

Conformational Differences between the Methoxy Groups of QA and QB Site Ubisemiquinones in Bacterial Reaction Centers: A Key Role for Methoxy Group Orientation in Modulating Ubiquinone Redox Potential

“…This arises from the polarization of the spin density distribution caused by the stronger hydrogen bonding to the O1 oxygen atom, as discussed previously. 38 …”

Probing the Menasemiquinone Binding Mode to Nitrate Reductase A by Selective 2H and 15N Labeling, HYSCORE Spectroscopy, and DFT Modeling

“…Assigning the detected 1 Ha nd 14 Nn ucleit oaparticular chemical group is not straightforward. For this purpose, various strategies have been used, including (i)uniform enrichment of the protein in 2 H [12][13][14] or 15 N [15][16][17][18][19][20][21][22] or of the solvent thankst oH 2 O/ 2 H 2 Oe xchange experiments; [12,[23][24][25][26][27][28] (ii)theoretical calculations of hyperfine and quadrupolar parameters, [13,[29][30][31][32][33] (iii)reconstitution experimentsu sing exogenous quinones with various substituents or chemically labeled with 2 H, [29,34] and (iv) comparison with spectroscopic data obtained on model SQs measured in organic solvents. [35][36][37][38][39] Ad irect and more physiological strategy relies on in vivo isotope labeling of individual amino acids or sets of amino acids,w hich,h owever,r equires engineered auxotrophics trains supplemented with isotopically enriched amino acids.…”