CHARACTERIZATION OF PHEOPHYTIN GROUND-STATES IN RHODOBACTER-SPHAEROIDES R26 PHOTOSYNTHETIC REACTION CENTERS FROM MULTISPIN PHEOPHYTIN ENRICHMENT AND 2-D C-13 MAS NMR DIPOLAR CORRELATION SPECTROSCOPY

The electronic ground states of pheophytin cofactors potentially invol ved in symmetry breaking between the A and B branch for electron trans port in the bacterial photosynthetic reaction center have been investi gated through a characterization of the electron densities at individu al atomic positions of pheophytin a from C-13 chemical shift data, A n ew experimental approach involving multispin C-13 labeling and 2-D NMR is presented. Bacterial photosynthetic reaction centers of Rhodobacte r sphaeroides R26 were reconstituted with uniformly C-13 biosynthetica lly labeled (plant) Pheo a in the two pheophytin binding sites. From t he multispin labeled samples 1-D and 2-D solid-state C-13 magic angle spinning NMR spectra could be obtained and used to characterize the ph eophytin a ground state in the Rb. sphaeroides R26 RCs, i.e., without a necessity for time-consuming selective labeling strategies involving organic synthesis. From the 2-D solid state C-13-C-13 correlation spe ctra collected with spinning speeds of 8 and 10 kHz, with mixing times of 1 and 0.8 ms, many C-13 resonances of the [U-C-13]Pheo a molecules reconstituted in the RCs could be assigned in a single set of experim ents. Parts of the pheophytins interacting with the protein, at the le vel of C-13 shifts modified by binding, could be identified. Small rec onstitution shifts are detected for the 17(2) side chain of ring IV. I n contrast, there is no evidence for electrostatic differences between the two Pheo a, for instance, due to a possibly strong selective elec trostatic interaction with Glu L104 on the active branch. The protonat ion states appear the same, and the NMR suggests a strong overall simi larity between the ground states of the two Pheo a, which is of intere st in view of the asymmetry of the electron transfer.