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 involved in symmetry breaking between the A and B branch for electron transport in the bacterial photosynthetic reaction center have been investigated through a characterization of the electron densities at individual atomic positions of pheophytin a from C-13 chemical shift data, A new experimental approach involving multispin C-13 labeling and 2-D NMR is presented. Bacterial photosynthetic reaction centers of Rhodobacter sphaeroides R26 were reconstituted with uniformly C-13 biosynthetically labeled (plant) Pheo a in the two pheophytin binding sites. From the 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 pheophytin 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 spectra 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 experiments. Parts of the pheophytins interacting with the protein, at the level of C-13 shifts modified by binding, could be identified. Small reconstitution shifts are detected for the 17(2) side chain of ring IV. In contrast, there is no evidence for electrostatic differences between the two Pheo a, for instance, due to a possibly strong selective electrostatic interaction with Glu L104 on the active branch. The protonation states appear the same, and the NMR suggests a strong overall similarity between the ground states of the two Pheo a, which is of interest in view of the asymmetry of the electron transfer.