FTIR spectroscopy shows weak symmetric hydrogen bonding of the QB carbonyl groups in Rhodobacter sphaeroides R26 reaction centres.

The absorption frequencies of the C = O and C = C (neutral state) and of the C...O (semiquinone state) stretching vibrations of QB have been assigned by FTIR spectroscopy, using native and site-specifically 1-, 2-, 3- and 4-13C-labelled ubiquinone-10 (UQ10) reconstituted at the QB binding site of Rhodobacter sphaeroides R26 reaction centres. Besides the main C = O band at 1641 cm-1, two smaller bands are observed at 1664 and 1651 cm-1. The smaller bands at 1664 and 1651 cm-1 agree in frequencies with the 1- and 4-C = O vibrations of unbound UQ10, showing that a minor fraction is loosely and symmetrically bound to the protein. The larger band at 1641 cm-1 indicates symmetric H-bonding of the 1- and 4-C = O groups for the larger fraction of UQ10 but much weaker interaction as for the 4-C = O group of QA. The FTIR experiments show that different C = O protein interactions contribute to the factors determining the different functions of UQ10 at the QA and the QB binding sites.

Asymmetric binding of the primary acceptor quinone in reaction centers of the photosynthetic bacterium Rhodobacter sphaeroides R26, probed with Q-band (35 GHz) EPR spectroscopy.

The reaction center (RC)-bound primary acceptor quinone QA of the photosynthetic bacterium Rhodobacter sphaeroides R26 functions as a one-electron gate. The radical anion QA.- is proposed to have an asymmetric electron distribution, induced by the protein environment. We replace the native ubiquinone-10 (UQ10) with specifically 13C-labelled UQ10, and use Q-band (35 GHz) EPR spectroscopy to investigate this phenomenon in closer detail. The direct observation of the 13C-hyperfine splitting of the gz-component of UQ10A.- in the RC and in frozen isopropanol shows that the electron spin distribution is symmetric in the isopropanol glass, and asymmetric in the RC. Our results allow qualitative assessment of the spin and charge distribution for QA.- in the RC. The carbonyl oxygen of the semiquinone anion nearest to the S = 2 Fe(2+)-ion and QB is shown to acquire the highest (negative) charge density.

13C magic angle spinning NMR characterization of the functionally asymmetric QA binding in Rhodobacter sphaeroides R26 photosynthetic reaction centers using site-specific 13C-labeled ubiquinone-10.

Photosynthetic reaction centers (RCs) of Rhodobacter sphaeroides R26 were reconstituted at the QA site with ubiquinone-10, selectively 13C-enriched on positions 1, 2, 3, 4, and 3-Me (IUPAC numbering). RCs dispersed in LDAO detergent were studied with 13C CP/MAS NMR spectroscopy at temperatures between 180 and 240 K, while RCs precipitated by removal of the detergent were investigated at ambient temperature and at temperatures down to 180 K. Electrostatic charge differences in QA induced by polarization from the protein are less than 0.02 electronic equivalent for any of the labeled positions. This includes the 4-carbonyl, which is therefore not significantly polarized by an electrostatic binding interaction with the protein. The QA site is slightly heterogeneous on the scale of the NMR as the observed line widths of the labels are between 150 and 300 Hz and inhomogeneous broadening is observed for the signals of positions 1, 2, and 3 upon cooling. This contrasts with earlier MAS observations for labels in the vicinity of the special pair. The chemical shifts are 184, 144, and 137 ppm for the labels at positions 1, 2, 3, and 12 ppm for the 3-methyl 13C. For the 4-carbonyl only at sample temperatures below approximately 255 K a CP/MAS response can be observed at 183 ppm. The principal components of the chemical shift tensors for the ring labels in QA were estimated using difference spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Asymmetric binding of the 1- and 4-C=O groups of QA in Rhodobacter sphaeroides R26 reaction centres monitored by Fourier transform infra-red spectroscopy using site-specific isotopically labelled ubiquinone-10.

Using 1-, 2-, 3- and 4-13C site-specifically labelled ubiquinone-10, reconstituted at the QA site of Rhodobacter sphaeroides R26 reaction centres, the infra-red bands dominated by the 1- and 4-C = O vibration of QA are assigned in the QA(-)-QA difference spectra. The mode dominated by the 4-C = O vibration is drastically downshifted in the reaction centres as compared with its absorption frequency in free ubiquinone-10. In contrast, the mode dominated by the 1-C = O vibration absorbs at similar frequencies in the free and the bound forms. The frequency shift of the 4-C = O vibration is due to a large decrease in bond order and indicates a strong interaction with the protein microenvironment in the ground state. In the charge-separated state the mode dominated by the semiquinone 4-C = O vibration is characteristic of strong hydrogen bonding to the microenvironment, whereas the mode dominated by the 1-C = O vibration indicates a weaker interaction. The asymmetric binding of the 1- and 4-C = O groups to the protein might contribute to the factors governing different redox reactions of ubiquinone-10 at the QA site as compared with its reactions at the QB site.