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 (15)N-labeled reaction centers, with the native high spin Fe(2+) exchanged for diamagnetic Zn(2+), prepared in (1)H(2)O and (2)H(2)O 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(δ)H···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 (13)C and (17)O carbonyl hyperfine couplings. The implications of these interactions for Q(B) function and comparisons with the Q(A) site are discussed.

Figure 1

Essential features of the Q_A and Q_B binding sites, showing putative hydrogen-bond donors to the quinone carbonyls. The structure is from 1dv3.pdb. The figure was prepared in VMD.

Figure 2

The HYSCORE spectra for protons H1 and H2 identified by ENDOR. Left column (A,C,E): contour presentation of the full cross-peaks. Right column B,D,F: calculated HYSCORE spectra using Q-band ENDOR derived hyperfine tensors shown in Tables 1 and 2, and time between first and second microwave pulses τ=136 ns. ¹H Zeeman frequency is 14.73 MHz (see also text). The spectra qualitatively demonstrate the relative intensity of different ridges. The wider and more extended ridges possess greater intensity.

Figure 3

Plots of cross-peaks from the calculated HYSCORE spectra of Figure 2 in the ν_α² vs. ν_β² coordinate system (see text for detailed explanations). The straight lines show the linear fit of plotted data points. The thick curved line is defined by (ν_α+ν_β)=2ν_H with proton Zeeman frequency 14.73 MHz. The dashed line corresponds to the diagonal line in the spectra.

Figure 4

Contour (A,B) and stacked (C,D) presentations of the experimental ¹H HYSCORE spectra of the Q_A SQ of Rba. sphaeroides reaction centers prepared in ¹H₂O and ²H₂O (magnetic field 345.9 mT (¹H₂O) and 346.0 mT (²H₂O), time between first and second pulses τ=136 ns, microwave frequency 9.702 GHz (¹H₂O) and 9.707 GHz (¹H₂O)). In the stacked presentation, in ¹H₂O, peak 3_A is hidden between ridges 1_A and 4_A.

Figure 5

Cross-peaks 1_A-4_A from ¹H HYSCORE spectrum in Figure 4 plotted in the ν_α² vs. ν_β² coordinate system (see text for detailed explanations). The straight lines show the linear fit of plotted data points. The thick curved line is defined by (ν_α+ν_β)=2ν_H with proton Zeeman frequency 14.73 MHz. The dashed line corresponds to the diagonal line in the spectra.

Figure 6

Contour (A,B) and stacked (C,D) presentations of the experimental ¹H HYSCORE spectra of the Q_B SQ of Rba. sphaeroides reaction centers prepared in ¹H₂O and ²H₂O. Spectra were obtained as a sum of three individual spectra recorded with time between first and second pulses τ=136, 200, and 400 ns, (magnetic field 345.1 mT (¹H₂O) and 344.6 mT (²H₂O), microwave frequency 9.680 GHz (¹H₂O) and 9.666 GHz (²H₂O)).

Figure 7

Cross-peaks 1_B-6_B from ¹H HYSCORE spectrum in Figure 6 plotted in the ν_α² vs. ν_β² coordinate system (see text for detailed explanations). The straight lines show the linear fit of plotted data points. The thick curved line is defined by (ν_α +ν_β)=2ν_H with proton Zeeman frequency 14.73 MHz. The dashed line corresponds to the diagonal line in the spectra.

Figure 8

Stacked presentations of sets of four-pulse ESEEM spectra of the Q_A and Q_B SQs in Rba. sphaeroides reaction centers prepared in ¹H₂O and ²H₂O. The spectra show the modulus of the Fourier transform along the time (T) axis for different times between first and second pulses, τ. The initial time τ is 100 ns in the farthest trace, and was increased by 12 ns in successive traces. Microwave frequency and magnetic field were 9.711 GHz and 346.2 mT (Q_A in ¹H₂O), 9.705 GHz and 346.0 mT (Q_A in ²H₂O). 9.708 GHz and 346.1 mT (Q_B in ¹H₂O), and 9.664 GHz and 344.5 mT (Q_B in ²H₂O).

Figure 9

Calculated Mulliken spin populations for the semiquinone in the Q_A and Q_B sites.