Co-crystallization and characterization of the photosynthetic reaction center-cytochrome c(2) complex from Rhodobacter sphaeroides

The photosynthetic reaction center (RC) of Rhodobacter sphaeroides and cytochrome c(2) (cyt c(2)), its physiological secondary electron donor, have been co-crystallized. The molar ratio of RC/cyt c(2) was found by SDS-PAGE and optical absorbance changes in the co-crystals to be 4. The crystals diffracted X-rays to 3.5 Angstrom. However, the resolution degraded during data collection. A data set, 82.5% complete, was collected to 4.5 Angstrom. The crystals belong to the tetragonal space group P4(3)2(1)2, with unit cell dimensions of a = b = 142.7 Angstrom and c = 254.8 Angstrom. The positions of the RCs in the unit cell were determined by molecular replacement. A comparable search for the cyt c(2) by this method was unsuccessful because of the small contribution of the cytochrome to the total scattering and because of its low occupancy. The cyt c(2) was positioned manually into patches of difference electron density, adjacent to the periplasmic surface of the M polypeptide subunit of the RC. The difference electron density was not sufficient for precise positioning of the cyt c(2), and its orientation was modeled by placing the exposed edge of the heme toward the primary donor of the reaction center D and by forming pairs for electrostatically interacting RC and cyt c(2) amino acid residues. The RC-cyt c(2) structure derived from the co-crystal data was supported by use of omit maps and structure refinement analyses. Cyt c(2) reduces the photooxidized primary donor D+ in 0.9 +/- 0.1 mu s in the co-crystals, which is the same as the fast electron transfer rate in vivo and in solution. This result provides strong evidence that the structure of the complex in the co-crystal is the same as in solution. Two additional methods were used to investigate the structure of the RC-cyt c(2) complex: (i) Docking calculations based on interprotein electrostatic interactions identified possible binding positions of the cyt c(2) on the RC. The cyt c(2) position with the lowest electrostatic energy is very similar to that of the cyt c(2) in the proposed co-crystal structure. (ii) Site-directed mutagenesis was used to modify two aspartic acid residues (M 184 and L155) on the periplasmic surface of the RC. Cyt c(2) binding affinity to these RCs and electron transfer rates to D+ in these RCs support the co-crystal structure of the RC-cyt c(2) complex.