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The three-dimensional structure of the cofactors of the reaction center of Rhodobacter sphaeroides R-26 has been determined by x-ray diffraction and refined at a resolution of 2.8 A with an R value of 26%. The main features of the structure are similar to the ones determined for Rhodopseudomonas viridis [Michel, H., Epp, 0. & Deisenhofer, J. (1986) EMBO J. 5, 2445EMBO J. 5, -2451. The cofactors are arranged along two branches, which are approximately related to each other by a 2-fold symmetry axis. The structure is well suited to produce light-induced charge separation across the membrane. Most of the structural features predicted from physical and biochemical measurements are confirmed by the x-ray structure.The reaction center (RC) is an integral membrane proteinpigment complex that mediates the primary processes of photosynthesis-i.e., the light-induced electron transfers from a donor to a series of acceptor species. The three-dimensional structure of the RC from the photosynthetic bacterium Rhodopseudomonas viridis has recently been determined by x-ray diffraction at a resolution of 2.9 A (1-3). In this paper, we report the structure analysis of the RC from another purple bacterium, the carotenoidless mutant R-26 of Rhodobacter sphaeroides (previously called Rhodopseudomonas sphaeroides). The motivation for undertaking the structure determination of the RC of a second bacterial species was 2-fold. (i) The RC from Rb. sphaeroides has been investigated for the past two decades and, consequently, is the best characterized RC (for reviews see refs. 4 and 5); in addition, the methodologies for manipulating its structure (e.g., exchanging cofactors, dissociating and reassociating the subunits) have been worked out in detail (4-10). (ii) The availability of structures from two organisms may help in elucidating structure-function relationships by correlating differences in structure with differences in function.The RC from Rb. sphaeroides is composed of three protein subunits-L, M, and H-and the following cofactors: four bacteriochlorophylls (Bchls), two bacteriopheophytins (Bphes), two ubiquinones, and one nonheme iron. The RC from R. viridis has an additional subunit, a cytochrome with four c-type hemes; its Bchls and Bphes are of the "b" type instead of the "a" type found in Rb. sphaeroides, and its primary quinone is a menaquinone. Notwithstanding these differences, the two structures were found to be very similar. This made it possible to use the method of molecular replacement (11) to solve the phase problem in the x-ray analysis (12)(13)(14). The crystals of Rb. sphaeroides diffract at least to a resolution of 2.6 A and retain the ability to perform the primary photochemistry (15). We have solved the structure of the protein and the cofactors to a resolution of 2.8 A with an R factor of 26%. In this paper, we report the structure of the cofactors. The structure of the protein, the relation of the RC protein to the membrane, and the interaction of the cofactors with the protein will be reported in ...

The effects of multiple changes in hydrogen bond interactions between the electron donor, a bacteriochlorophyll dimer, and htdine residues in the reaction center from Rhodobacter sphaeroides have been igaed. Sitedirected mutations were deiged to add or remove hydrogen bonds between the 2-acetyl groups of the dimer and istidine residues at the setry-related sites Hls-L168 and Phe-M197, and between the 9-keto groups and Leu-L131 and Leu-M160. The addition of a hydrogen bond was correlated with an increase in the dimer midpoint potential. Measurements on double and triple mutants showed that changes In the midpoint potential due to alterations at the individual sites were additive.Midpoint potentials ranging from 410 to 765 mV, compared with 505 mV for wild type, were achieved by various combinations of mutations. The optical absorption spectra of the reaction centers showed relatively minor changes in the position of the donor absorption band, indicating that the addiion of hydrogen bonds to hide pimril destabilized the oxidized state of the donor and had little effect on the excited state relative to the ground state. Despite the change in energy of the charge-separated states by up to 260 meV, the mutant reaction centers were still capable of electron taner to the primary quinone. The increase in midpoint potential was correlated with an increase in the rate of charge recombination from the primary quin , and a fit of these data using the Marcus equation idicated that the reorgn i energy for this reaction is =400 meV higher than the change in free energy in wild type. The mutants were still capable of photosynthetic growth, although at reduced rates relative to the wild type. These results suggest a role for protein-cofactor interactions-n particular, hididonor interactions-In establishing the redox potentials needed for electron transfer in biological systems.Although the oxidation-reduction midpoint potentials of identical cofactors in redox proteins can vary by several hundred millivolts, the specific interactions of the cofactor with the protein that result in the variation in midpoint potential are not well understood. The primary electron donor in reaction centers from the purple photosynthetic bacterium Rhodobacter sphaeroides is a bacteriochlorophyll (Bchl) dimer designated P (reviewed in refs. 1-3). The two Bchls of the dimer, labeled A and B, overlap in ring I, where they are separated by -3.5 A. The midpoint potential of the primary donor is =500 mV in wild-type reaction centers from Rb. sphaeroides (4-6) and is expected to be a critical parameter for electron transfer reactions that involve the donor, as alteration of the potential will result in a change in the driving force for these reactions.Mutagenesis experiments have shown that hydrogen bonds between histidine residues and the conjugated carbonyls of the Bchls in the dimer can alter the midpoint potential by significant amounts (5, 7-10). For each Bchl there are two groups, the 9-keto group of ring V and the 2-acetyl group of ring I, t...

The three-dimensional structure of the protein subunits of the reaction center (RC) of Rhodobacter sphaeroides has been determined by x-ray diffraction at a resolution of 2.8 A with an R factor of 26%. The L and M subunits each contain five transmembrane helices and several helices that do not span the membrane. The L and M subunits are related to each other by a 2-fold rotational symmetry axis that is approximately the same as that determined for the cofactors. The H subunit has one transmembrane helix and a globular domain on the cytoplasmic side, which contains a helix that does not span the membrane and several 13-sheets. The structural homology with RCs from other purple bacteria is discussed. A structure of the complex formed between the water soluble cytochrome c2 and the RC from Rb. sphaeroides is proposed.The reaction center (RC) from photosynthetic bacteria is an integral membrane protein complex, which contains a number of cofactors that mediate the primary photochemistry. The RC of Rhodobaccter sphaeroides is composed of three protein subunits L, M, and H having 281, 307, and 260 residues, respectively (1-3). From an analysis of the amino acid sequence of the subunits, five membrane-spanning helices in both the L and M subunits and one in the H subunit were predicted (1-3). Furthermore, labeling experiments showed that the RC spans the membrane with the bulk of the H subunit on the cytoplasmic side; the amino terminus of L was determined to be on the cytoplasmic side (for reviews, see refs. 4 and 5).The three-dimensional structure of the RC has been obtained to a resolution of 2.8 A. In a previous paper, we described the x-ray diffraction analysis and reported the structure of the cofactors (6). In this paper, we present the structure of the individual subunits as well as the entire RC complex. A structure of the complex between the RC and the water soluble cytochrome c, is proposed. The overall structure and homology with other RCs, in particular that of Rhodopseudomonas viridis (7), is discussed. METHODS RC crystals having the space group P212121 were analyzed by x-ray diffraction as described (6). The structure ofthe RC was determined at a resolution of 2.8 A with an R factor of 26% between calculated and observed structure factors. The secondary structure was identified on the basis of main-chain hydrogen bonding and torsion angle patterns. Due to deviations from ideal geometry, the boundaries of the regions of a-helices and a-sheets have an uncertainty of several residues. Final assignments of the boundaries were checked visually by computer graphics [Evans and Sutherland PS 300 with FRODO program (8)].The directions of the helical axes were equated with the normals of the greatest square plane fit of the Ca atoms of each helix (9). The radius of curvature to an a-helix was calculated by two methods: (i) from the radius of the best circle fit to the projections of Ca atoms onto the helical axis (10); and (ii) from the directions of the helical axes based on residues in the first and last...

The initial electron transfer dynamics during photosynthesis have been studied in Rhodobacter sphaeroides reaction centers from wild type and 14 mutants in which the driving force and the kinetics of charge separation vary over a broad range. Surprisingly, the protein relaxation kinetics, as measured by tryptophan absorbance changes, are invariant in these mutants. By applying a reaction-diffusion model, we can fit the complex electron transfer kinetics of each mutant quantitatively, varying only the driving force. These results indicate that initial photosynthetic charge separation is limited by protein dynamics rather than by a static electron transfer barrier.