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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.

The three-dimensional structure of the reaction center (RC) from Rhodobacter sphaeroides has been determined by x-ray diffraction to a resolution of 2.8 A with an R value of 24%. The interactions of the protein with the primary quinone, QA, secondary quinone, QB and the nonheme iron are described and compared to those of RCs from Rhodopseudomonas viridis. Structural differences between the QA and QB environments that contribute to the function of the quinones (the electron transfer from QA to QB and the charge recombination of Q-, Q-with the primary donor) are delineated. The protein residues that may be involved in the protonation of QB are identified. A pathway for the doubly reduced QB to dissociate from the RC is proposed. The interactions between QB and the residues that have been changed in herbicide-resistant mutants are described. The environment of the nonheme iron is compared to the environments of metal ions in other proteins.The reaction center (RC) is a protein-pigment complex that mediates the primary photochemistry of photosynthetic systems. The RC from Rhodobacter sphaeroides R-26 is composed of three protein subunits (L, M, and H) and several cofactors (for a review, see ref. 1). Serving as electron acceptors are the primary quinone QA and the secondary quinone QB. In Rb. sphaeroides both quinones are ubiquinones, whereas in Rhodopseudomonas viridis QA is a menaquinone and QB is a ubiquinone.The three-dimensional structure of the RC from Rb. sphaeroides R-26 has been obtained to a resolution of2.8 A (2-4) and an R value of 24%. In this paper, we describe the interactions of the protein with the quinones and the nonheme iron. We emphasize structural features that appear to be important in electron and proton transfer. Differences in the interactions of the protein with the quinones in Rb. sphaeroides and Rps. viridis are described, although a detailed comparison between the QB interactions is difficult because of the low occupancy of the QB sites in crystals ofRps. viridis (5, 6). The interactions with the nonheme iron are found to be very similar for the two bacterial species. The interactions ofthe protein with the other cofactors as well as the crystallographic procedures have been reported (7). In a subsequent paper (8), the asymmetries ofthe two branches of the cofactors and a comparison of RCs from different species will be presented. RESULTSThe Primary Quinone. The primary quinone, QAI is located near the cytoplasmic side of the RC. Its position and orientation are well defined by the electron density, although there is no steric hindrance to moving the quinone to a position indicated by the dotted line in Fig. 1 (see below). The normal of the quinone ring forms an angle of =60°with respect to the membrane normal as defined by the direction of the twofold symmetry axis (4). QA interacts with the amino acid residues of the D, de, and E helices of the M subunit (3) (see Fig. 1).The two carbonyl oxygens Of QA are within hydrogenbonding distance to the peptide nitrogen of Ala M260 and ...

Mutations were made in four residues near the bacteriochlorophyll cofactors of the photosynthetic reaction center from Rhodobacter sphaeroides. These mutations, L131 Leu to His and M160 Leu to His, near the dimer bacteriochlorophylls, and M203 Gly to Asp and L177 Ile to Asp, near the monomer bacteriochlorophylls, were designed to result in the placement of a hydrogen bond donor group near the ring V keto carbonyl of each bacteriochlorophyll. Perturbations of the electronic structures of the bacteriochlorophylls in the mutants are indicated by additional resolved transitions in the bacteriochlorophyll absorption bands in steady-state low-temperature and time-resolved room temperature spectra in three of the resulting mutant reaction centers. The major effect of the two mutations near the dimer was an increase up to 80 mV in the donor oxidation-reduction midpoint potential. Correspondingly, the calculated free energy difference between the excited state of the primary donor and the initial charge separated state decreased by up to 55 mV, the initial forward electron-transfer rate was up to 4 times slower, and the rate of charge recombination between the primary quinone and the donor was approximately 30% faster in these two mutants compared to the wild type. The two mutations near the monomer bacteriochlorophylls had minor changes of 25 mV or less in the donor oxidation-reduction potential, but the mutation close to the monomer bacteriochlorophyll on the active branch resulted in a roughly 3-fold decrease in the rate of the initial electron transfer.

The three-dimensional structures of the cofactors and protein subunits of the reaction center (RC) from the carotenoidless mutant strain of Rhodobacter sphaeroides R-26 and the wild-type strain 2.4. The reaction center (RC) is an integral membrane proteinpigment complex that mediates the primary processes of photosynthesis-i.e., the light-induced electron transfer from a donor to a series of acceptors. reported the structure of the cofactors (3), protein subunits (5), and the membrane-protein interactions (6) ofthe RC from Rb. sphaeroides R-26. In this paper, we discuss the interactions of the proteins with the cofactors D, B, and 4, the carotenoid, C, and the detergent f3-octyl glucoside (J30G).The protein interactions involving the quinones and iron will be reported in a subsequent publication (7). The structure of the RC from Rb. sphaeroides is compared with that of Rhodopseudomonas viridis (8), with special emphasis on the structural features that are believed to be important in the electron transfer process. It should be noted that only the structure of the unexcited state of the RC has been reported (2, 8, 9); conformation changes accompanying the absorption of light and subsequent charge separation have not yet been reported. EXPERIMENTAL PROCEDURESRC from Rb. sphaeroides R-26. Data collection, processing, and initial refinement of the structure of the RC from Rb. sphaeroides R-26 at 2.8 A resolution has been described (3).Subsequent rebuilding of the atomic structure was facilitated by the use of modified "omit-maps." These are typically calculated by removing all atoms from a specified volume in the unit cell and then using the remaining atoms to phase the Fourier calculation for electron density in that volume (10). This approach tends to minimize bias in the electron density map toward the current atomic model. In the present work, a least-squares refinement of an initial omit-map was implemented. The electron density map was sampled at 144, 96, and 144 grid points along the crystallographic a, b, and c axes, respectively. Electron density values within a specific block of 12 x 12 x 12 grid points were varied, while grid points outside this block were fixed at the electron density of the current model. Values of the electron density within the block were refined by a least-squares calculation to minimize the difference between the magnitudes of observed and calculated structure factors. This procedure was repeated for all 288 blocks in the crystallographic asymmetric unit. An asymmetric unit was reconstructed from the blocks, followed by a least-squares version of solvent flattening. The quality of the resulting electron density maps was significantly improved relative to (IF01 -IFJI) or (21F01 -IFJI) maps, which are often used in model building. After adjustments of the cofactors, the residues in the L and M subunits, and the addition of two POG molecules, the atomic structure was refined to an R factor of 24.0%, with the restrained least-