Biochemistry zyxwvuts 1995,34, zyxwvu 10229- 10236 10229 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- 1 zyx Ot W. B. S. van Liemt,t G. J. Boender,; P. Gast,§ A. J. Hoff,§ J. Lugtenburg,$ and H. J. M. de Groot*q$ Leiden Institute of Chemistry, Gorlaeus Laboratories, and Department of Biophysics, zyxwv Huygens Laboratorium, Leiden University, zyxwvut P.O. Box 9502, 2300 RA Leiden, The Netherlands Received January 6, 1995; Revised Manuscript Received June 2, 1995@ ABSTRACT: Photosynthetic reaction centers (RCs) of Rhodobacter sphaeroides R26 were reconstituted at the QA site with ubiquinone-10, selectively I3C-enriched on positions 1, 2, 3, 4, and 3-Me (IUPAC numbering). RCs dispersed in LDAO detergent were studied with I3C 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 I3C. For the 4-carbonyl only at sample temperatures below -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. Although the asymmetry of the anisotropy of the 4-I3C signal from QA is only moderately different from the anisotropy of the 4 position in crystalline UQlo, it is concluded that the NMR is compatible with a decrease of the 4 C=O bond order upon binding to the protein. The temperature-dependent asymmetry between the two carbonyls in QA indicates that the putative strong interaction with the protein at position 4 involves dynamic character, which may be of importance to the specific QA redox chemistry. The photosynthetic RC' of Rhodobacter (Rb.) sphaeroides R26 is a transmembrane protein complex that consists of three polypeptide chains (L, M, and H) and nine cofactors: two ubiquinones- 10, four bacteriochlorophylls, two bacte- riopheophytins, and one non-heme Fe2+. The cofactors form two branches, designated A and B, with a nearly 2-fold symmetry (Rees et al., 1989; Chang et al., 1991; Ermler et al., 1994). The ubiquinone residing in the A branch is denoted by QA and is very tightly bound. It permanently occupies the reaction center during the photochemistry. Upon illumination of the protein complex an electron is transported from a bacteriochlorophyll pair (P) at the periplasmic side of the membrane to the primary quinone QA [for a review, see, e.g., Bixon et al., (1991)l. Interest- zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA + This research was supported by the Netherlands Foundations for Biophysical and Chemical Research and financed by the Netherlands Organisation for Scientific Research (NWO). P.G and H.J.M.d.G. have received a research career development fellowship from the Koninklijke Nederlandse Academie van Wetenschappen (Royal Dutch Academy of Sciences). * To whom correspondence should be addressed. zyxwvuts 4 Gorlaeus Laboratories. zyxwvutsrq 5 Huygens Laboratory. @Abstract published in Advance ACS Abstracts, July 15, 1995. Abbreviations: CP, cross-polarization: CSA, chemical shift ani- sotropy; DQo, duroquinone: FWHM, full width at half-maximum; LDAO, NAN-dimethyldodecylamine-N-oxide: MAS, magic angle spin- ning: NMR, nuclear magnetic resonance; Qo, benzoquinone: RC, reaction center: RT, room temperature; TMS, tetramethylsilane; UQi0, ubiquinone- 10. 0006-2960/95/0434- 10229$09.00/0 ingly, QA temporarily takes on only one electron under physiological conditions, thereby reaching the semiquinone state (Slooten, 1972). This is rather remarkable, as quinones generally undergo two-step reduction to the corresponding quinoles (Morrison et al., 1982). Therefore, specific protein- cofactor interactions should be responsible for the QAredox propexties. In addition, the tight binding of QA in the RC can also be attributed to interactions with the protein environment. Protein-QA interactions have been the subject of extensive investigations. For instance, at an early stage UV/Vis spectroscopy has been used to identify ubiquinone as the primary electron acceptor and to determine the protonation state of the semiquinone (Slooten, 1972; Wraight, 1979; VermCglio, 1982). In the X-ray structures of the QA binding site, the UQlo carbonyls have different distances to possible protein hydrogen-bond donors, indicating a functional asym- metry between these positions (Allen et al., 1987; Chang et al., 1991; Ermler et al., 1994). Functional asymmetry also transpired from FTIR studies on the QAbinding site and was inferred from the examination of the binding of model compounds in the QA site (Breton et al., 1994a,b; Brudler et al., 1994; Gunner et al., 1985; Warncke & Dutton, 1993). In addition, from ENDOR measurements and from recent Q-band EPR spectroscopy on QA*-, a pronounced asymmetry between both carbonyl positions in the semiquinone state was observed (Lubitz et al., 1985; Feher et al., 1985; Van den Brink et al., 1994). zyxwvutsrqponmlkjihgfedcbaZYXWVUT 0 1995 American Chemical Society