C-13 MAGIC-ANGLE-SPINNING NMR CHARACTERIZATION OF THE FUNCTIONALLY ASYMMETRIC Q(A) BINDING IN RHODOBACTER-SPHAEROIDES R26 PHOTOSYNTHETIC REACTION CENTERS USING SITE-SPECIFIC C-13-LABELED UBIQUINONE-L0

Photosynthetic reaction centers (RCs) of Rhodobacter sphaeroides R26 w ere reconstituted at the Q(A) site with ubiquinone-10, selectively C-1 3-enriched on positions 1, 2, 3, 4, and 3-Me (IUPAC numbering). RCs di spersed in LDAO detergent were studied with C-13 CP/MAS NMR spectrosco py at temperatures between 180 and 240 K, while RCs precipitated by re moval of the detergent were investigated at ambient temperature and at temperatures down to 180 K. Electrostatic charge differences in Q(A) induced by polarization from the protein are less than 0.02 electronic equivalent for any of the labeled positions. This includes the 4-carb onyl, which is therefore not significantly polarized by an electrostat ic binding interaction with the protein. The Q(A) site is slightly het erogeneous on the scale of the NMR as the observed line widths of the labels are between 150 and 300 Hz and inhomogeneous broadening is obse rved for the signals of positions 1, 2, and 3 upon cooling. This contr asts with earlier MAS observations for labels in the vicinity of the s pecial pair. The chemical shifts are 184, 144, and 137 ppm for the lab els at positions 1, 2, 3, and 12 ppm for the S-methyl C-13. For the 4- carbonyl only at sample temperatures below similar to 255 K a CP/MAS r esponse can be observed at 183 ppm. The principal components of the ch emical shift tensors for the ring labels in Q(A) were estimated using difference spectroscopy. Although the asymmetry of the anisotropy of t he 4-C-13 signal from Q(A) is only moderately different from the aniso tropy of the 4 position in crystalline UQ(10), it is concluded that th e NMR is compatible with a decrease of the 4 C=O bond order upon bindi ng to the protein. The temperature-dependent asymmetry between the two carbonyls in Q(A) indicates that the putative strong interaction with the protein at position 4 involves dynamic character, which may be of importance to the specific Q(A) redox chemistry.