Structure of the reaction center from Rhodobacter sphaeroides R-26: the cofactors.

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, O. & Deisenhofer, J. (1986) EMBO J. 5, 2445-2451]. 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.     

Stark spectroscopy of the Rhodobacter sphaeroides reaction center heterodimer mutant.

The effect of an electric field has been measured on the absorption spectrum (Stark effect) of the heterodimer mutant (M)H202L of Rhodobacter sphaeroides reaction centers, where the primary electron donor consists of one bacteriochlorophyll alpha and one bacteriopheophytin alpha. The electronic absorption spectrum of the heterodimer mutant from 820-950 nm is relatively featureless in a poly(vinyl alcohol) film, but it exhibits some structure in a glycerol/water glass at 77 K. A feature is seen in the Stark effect spectrum of the heterodimer at 77 K centered at 927 and 936 nm in poly(vinyl alcohol) and a glycerol/water glass, respectively. This feature has approximately the same shape and width as the Stark effect for the primary electron donor of the wild type, which consists of a pair of bacteriochlorophyll alpha molecules. The angle zeta A between the transition moment at the frequency of absorption and the difference dipole delta muA is 36 +/- 2 degrees in the wild type and 32 +/- 2 degrees for that feature in the heterodimer. A range of values for [delta muA] = (13-17)/f Debye units (where f is the local field correction) is obtained for the 936-nm feature in glycerol/water, depending on analysis method. This feature is interpreted as arising from a transition to the lower exciton state of the heterodimer, which is more strongly mixed with a low-lying charge transfer transition than in the wild type.     

Electron transfer in the Rhodobacter sphaeroides reaction center assembled with zinc bacteriochlorophyll

The cofactor composition and electron-transfer kinetics of the reaction center (RC) from a magnesium chelatase (bchD) mutant of Rhodobacter sphaeroides were characterized. In this RC, the special pair (P) and accessory (B) bacteriochlorophyll (BChl) -binding sites contain Zn-BChl rather than BChl a. Spectroscopic measurements reveal that Zn-BChl also occupies the H sites that are normally occupied by bacteriopheophytin in wild type, and at least 1 of these Zn-BChl molecules is involved in electron transfer in intact Zn-RCs with an efficiency of >95% of the wild-type RC. The absorption spectrum of this Zn-containing RC in the near-infrared region associated with P and B is shifted from 865 to 855 nm and from 802 to 794 nm respectively, compared with wild type. The bands of P and B in the visible region are centered at 600 nm, similar to those of wild type, whereas the H-cofactors have a band at 560 nm, which is a spectral signature of monomeric Zn-BChl in organic solvent. The Zn-BChl H-cofactor spectral differences compared with the P and B positions in the visible region are proposed to be due to a difference in the 5th ligand coordinating the Zn. We suggest that this coordination is a key feature of protein–cofactor interactions, which significantly contributes to the redox midpoint potential of H and the formation of the charge-separated state, and provides a unifying explanation for the properties of the primary acceptor in photosystems I (PS1) and II (PS2).     

Structure of the reaction center from Rhodobacter sphaeroides R-26: membrane-protein interactions.

The energetics of membrane-protein interactions are analyzed with the three-dimensional model of the photosynthetic reaction center (RC) from Rhodobacter sphaeroides. The position of the RC in the membrane and the thickness of the membrane were obtained by minimizing the hydrophobic energy with the energy function of Eisenberg and McLachlan. The 2-fold symmetry axis that relates the L and M subunits is, within the accuracy of 5 degrees, parallel to the normal of the membrane. The thickness of the membrane is estimated to be 40-45 A. Residues that are exposed to the membrane are relatively poorly conserved in the sequences of homologous RC proteins. The surface area of the RC is comparable to the surface areas of water-soluble proteins of similar molecular weight. The volumes of interior atoms in the RC are also similar to those of water-soluble proteins, indicating the same compact packing for both types of proteins. The electrostatic potential of the cofactors was calculated. The results show an asymmetry in the potential between the two possible pathways of electron transfer, with the A branch being preferred electrostatically.     

Structure of the reaction center from Rhodobacter sphaeroides R-26: the protein subunits.

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 beta-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.     

Multiple pathways for ultrafast transduction of light energy in the photosynthetic reaction center of Rhodobacter sphaeroides

A pathway of electron transfer is described that operates in the wild-type reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides. The pathway does not involve the excited state of the special pair dimer of bacteriochlorophylls (P*), but instead is driven by the excited state of the monomeric bacteriochlorophyll (BA*) present in the active branch of pigments along which electron transfer occurs. Pump-probe experiments were performed at 77 K on membrane-bound RCs by using different excitation wavelengths, to investigate the formation of the charge separated state P+HA-. In experiments in which P or BA was selectively excited at 880 nm or 796 nm, respectively, the formation of P+HA- was associated with similar time constants of 1.5 ps and 1. 7 ps. However, the spectral changes associated with the two time constants are very different. Global analysis of the transient spectra shows that a mixture of P+BA- and P* is formed in parallel from BA* on a subpicosecond time scale. In contrast, excitation of the inactive branch monomeric bacteriochlorophyll (BB) and the high exciton component of P (P+) resulted in electron transfer only after relaxation to P*. The multiple pathways for primary electron transfer in the bacterial RC are discussed with regard to the mechanism of charge separation in the RC of photosystem II from higher plants.     

Structure of the reaction center from Rhodobacter sphaeroides R-26: protein-cofactor (quinones and Fe2+) interactions.

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 QA-, QB- 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.     

Key role of proline L209 in connecting the distant quinone pockets in the reaction center of Rhodobacter sphaeroides

Photosynthetic bacterial reaction centers convert light excitation into chemical free energy. The initial electron transfer leads to the consecutive semireductions of the primary (Q(A)) and secondary (Q(B)) quinone acceptors. The Q(A)(-) and Q(B)(-) formations induce proton uptake from the bulk. Their magnitudes (H(+)/Q(A)(-) and H(+)/Q(B)(-), respectively) probe the electrostatic interactions within the complex. The pH dependence of H(+)/Q(A)(-) and H(+)/Q(B)(-) were studied in five single mutants modified at the L209 site (L209P-->F,Y,W,E,T). This residue is situated at the border of a continuous chain of water molecules connecting Q(B) to the bulk. In the wild type (WT), a proton uptake band is present at high pH in the H(+)/Q(A)(-) and H(+)/Q(B)(-) curves and is commonly attributed to a cluster of acidic groups situated nearby Q(B). In the H(+)/Q(A)(-) curves of the L209 variants, this band is systematically absent but remains in the H(+)/Q(B)(-) curves. Moreover, notable increase of H(+)/Q(B)(-) is observed in the L209 mutants at neutral pH as compared with the WT. The large effects observed in all L209 mutants are not associated with significant structural changes (Kuglstatter, A., Ermler, U., Michel, H., Baciou, L. & Fritzsch, G. Biochemistry (2001) 40, 4253-4260). Our data suggest that, in the L209 mutants, the Q(B) cluster does not respond to the Q(A)(-) formation as observed in the WT. We propose that, in the mutants, removal of the rigid proline L209 breaks a necessary hydrogen bonding connection between the quinone sites. These findings suggest an important role for structural rigidity in ensuring a functional interaction between quinone binding sites.     

Femtosecond coherent transient infrared spectroscopy of reaction centers from Rhodobacter sphaeroides.

Protein and cofactor vibrational dynamics associated with photoexcitation and charge separation in the photosynthetic reaction center were investigated with femto-second (300-400 fs) time-resolved infrared (1560-1960 cm-1) spectroscopy. The experiments are in the coherent transient limit where the quantum uncertainty principle governs the evolution of the protein vibrational changes. No significant protein relaxation accompanies charge separation, although the electric field resulting from charge separation modifies the polypeptide carbonyl spectra. The potential energy surfaces of the "special pair" P and the photoexcited singlet state P* and environmental perturbations on them are similar as judged from coherence transfer measurements. The vibrational dephasing time of P* modes in this region is 600 fs. A subpicosecond transient at 1665 cm-1 was found to have the kinetics expected for a sequential electron transfer process. Kinetic signatures of all other transient intermediates, P, P*, and P+, participating in the primary steps of photosynthesis were identified in the difference infrared spectra.     

Structure of the reaction center from Rhodobacter sphaeroides R-26 and 2.4.1: symmetry relations and sequence comparisons between different species.

Photosynthetic reaction centers from purple bacteria exhibit an approximate twofold symmetry axis, which relates both the cofactors and the L and M subunits. For the reaction center from Rhodobacter sphaeroides, deviations from this twofold symmetry axis have been quantitated by superposing, by a 180 degrees rotation, the cofactors of the B branch onto the A branch and the M subunit onto the L subunit. An alignment of the sequences of the L and M subunits from four purple bacteria, one green bacterium, and the D1 and D2 subunits of a photosystem II-containing green alga is presented. The residues that are conserved in all six species are shown in relation to the structure of Rb. sphaeroides and their possible role in the function of the reaction center is discussed. A method is presented for characterizing the exposure of alpha-helices to the membrane based on the periodicity of conserved residues. This method may prove useful for modeling the three-dimensional structures of membrane proteins.     

Crystallization of reaction center from Rhodopseudomonas sphaeroides: preliminary characterization.

Reaction centers (RCs), integral membrane proteins that mediate the conversion of light into chemical energy, were crystallized by two different vapor diffusion techniques. In one method, small amphipathic molecules (1,2,3-heptanetriol and triethylammonium phosphate) were added to the RCs that had been solubilized in detergent. In the second method, crystallization occurred near the phase boundaries of a two-phase system created by the addition of polyethylene glycol and NaCl to RCs in octyl beta-D-glucoside. Several different crystal forms were obtained; two were analyzed by x-ray diffraction. One was monoclinic (space group P2) with beta = 105 degrees, and a = 70 A, b = 105 A, and c = 85 A, two RCs per unit cell, and one RC per asymmetric unit; the crystal diffracted to 3.5 A at 17 degrees C. The other crystal form was orthorhombic (space group C222) with a = 185 A, b = 170 A, and c = 105 A, with eight RCs per unit cell and one RC per asymmetric unit. Reversible light-induced EPR signals of the primary donor (bacteriochlorophyll dimer) showed that the RCs in the crystal were fully active. From the angular dependence of the EPR signal the molecular g anisotropy of the bacteriochlorophyll dimer was deduced to be g perpendicular - g parallel = (64 +/- 3) X 10(-5). Linear dichroism measurements were performed on the monoclinic crystal. The two bands at 535 and 544 nm assigned to the Qx transitions of the bacteriopheophytins were resolved and preliminary orientations of some of the pigments were obtained.     

Specific alteration of the oxidation potential of the electron donor in reaction centers from Rhodobacter sphaeroides.

The effects of multiple changes in hydrogen bond interactions between the electron donor, a bacteriochlorophyll dimer, and histidine residues in the reaction center from Rhodobacter sphaeroides have been investigated. Site-directed mutations were designed to add or remove hydrogen bonds between the 2-acetyl groups of the dimer and histidine residues at the symmetry-related sites His-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 addition of hydrogen bonds to histidines primarily 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 transfer to the primary quinone. The increase in midpoint potential was correlated with an increase in the rate of charge recombination from the primary quinone, and a fit of these data using the Marcus equation indicated that the reorganization energy for this reaction is approximately 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--in particular, histidine-donor interactions--in establishing the redox potentials needed for electron transfer in biological systems.     

The Stark effect in reaction centers from Rhodobacter sphaeroides R-26 and Rhodopseudomonas viridis.

The effect of an electric field on the optical absorption (Stark effect) of reaction centers (RCs) from Rhodobacter sphaeroides and Rhodopseudomonas viridis embedded in films of poly(vinyl alcohol) was measured. The infrared bands were investigated at 295 K and 77 K. In RCs from Rp. viridis at 77 K six peaks (at 982, 849, 835, 818, 803, and 787 nm), associated with the Qy transitions of the six pigments, were resolved; in addition, a small broad band at 865 nm was resolved. In RCs from Rb. sphaeroides only five bands (at 877, 817, 802, 761, and 754 nm) assigned to the Qy transitions were resolved; in addition, two small bands at 697 and 683 nm were observed. The additional bands have been tentatively assigned to vibrational side bands, although the contribution from charge-transfer states cannot be excluded. The Stark spectra had line shapes similar to the second derivative of the absorption spectra and were interpreted in terms of the interaction between the applied electric field and the dipole moments of the ground and excited states. Analyses of the spectra yielded the apparent change in dipole moment delta mu app = f delta mu (where the factor f corrects for the difference between the local field and the applied field) and delta, the angle between delta mu---- and the transition moment mu trans. At 77 K the values of delta mu----app and delta for the peaks at 877, 802, and 761 nm in Rb. sphaeroides were 6.5 debye, 38 degrees; 2.1 debye, 23 degrees; and 3.5 debye, 8 degrees. In Rp. viridis the debye values for the peaks at 982, 835, 818, and 787 were 8.2, 40 degrees; 1.8, 50 degrees; 3.4, 14 degrees; and 2.7, 0 degrees. The large values of delta mu app associated with the long-wavelength peak of the bacteriochlorophyll dimers are consistent with a significant charge-transfer contribution to the excited state of the primary donor.     

Primary structure of the M subunit of the reaction center from Rhodopseudomonas sphaeroides

The reaction center is a membrane-bound bacteriochlorophyll-protein complex that mediates the primary photochemical events in the photosynthetic bacterium Rhodopseudomonas sphaeroides. The previously determined amino-terminal sequences of the three subunits of the reaction center protein were used to design synthetic mixed oligonucleotide probes for the structural genes encoding the subunits. One of these probes was used to isolate and clone a fragment of DNA from R. sphaeroides that contained the gene encoding the M subunit. The nucleotide sequence of this gene was determined by the dideoxy method. In addition, a number of tryptic and chymotryptic peptides from the M protein were isolated and subjected to sequence analysis, and the sequence of the carboxyl terminus was determined. Together with the amino-terminal sequence, the data establish the primary structure of the M protein. The distribution of hydrophobic residues in the amino acid sequence suggests the presence of five membrane-spanning segments. A significant homology was found between the amino acid sequence of the M subunit and a thylakoid membrane protein (M_r 32,000) from spinach that has been implicated in herbicide and quinone binding.     

Structure of the reaction center from Rhodobacter sphaeroides R-26 and 2.4.1: protein-cofactor (bacteriochlorophyll, bacteriopheophytin, and carotenoid) interactions.

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 2.4.1 have been determined by x-ray diffraction to resolutions of 2.8 A and 3.0 A with R values of 24% and 26%, respectively. The bacteriochlorophyll dimer (D), bacteriochlorophyll monomers (B), and bacteriopheophytin monomers (phi) form two branches, A and B, that are approximately related by a twofold symmetry axis. The cofactors are located in hydrophobic environments formed by the L and M subunits. Differences in the cofactor-protein interactions between the A and B cofactors, as well as between the corresponding cofactors of Rb, sphaeroides and Rhodopseudomonas viridis [Michel, H., Epp, O. & Deisenhofer, J. (1986) EMBO J. 3, 2445-2451], are delineated. The roles of several structural features in the preferential electron transfer along the A branch are discussed. Two bound detergent molecules of beta-octyl glucoside have been located near BA and BB. The environment of the carotenoid, C, that is present in RCs from Rb. sphaeroides 2.4.1 consists largely of aromatic residues of the M subunit. A role of BB in the triplet energy transfer from D to C and the reason for the preferential ease of removal of BB from the RC is proposed.     

Stark effect spectroscopy of Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centers

The nature of the initially excited state of the primary electron donor or special pair has been investigated by Stark effect spectroscopy for reaction centers from the photosynthetic bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides at 77 K. The data provide values for the magnitude of the difference in permanent dipole moment between the ground and excited state, [unk]Δμ[unk], and the angle [unk] between Δμ and the transition dipole moment for the electronic transition. [unk]Δμ[unk] and [unk] for the lowest-energy singlet electronic transition associated with the special pair primary electron donor were found to be very similar for the two species. [unk]Δμ[unk] for this transition is substantially larger than for the Q_y transitions of the monomeric pigments in the reaction center or for pure monomeric bacteriochlorophylls, for which Stark data are also reported. We conclude that the excited state of the special pair has substantial charge-transfer character, and we suggest that charge separation in bacterial photosynthesis is initiated immediately upon photoexcitation of the special pair. Data for Rhodobacter sphaeroides between 340 and 1340 nm are presented and discussed in the context of the detection of charge-transfer states by Stark effect spectroscopy.     

Primary structure of the L subunit of the reaction center from Rhodopseudomonas sphaeroides.

The reaction center is an integral membrane protein that, together with several cofactors, mediates the primary photochemical events in bacterial photosynthesis. The amino-terminal sequences of the three subunits, L, M, and H, of the reaction center protein and the sequence of the structural gene encoding the M subunit have been reported previously. In the present study, we found that the 3' end of the structural gene encoding the L subunit overlaps by eight bases the 5' end of the gene encoding the M subunit. The primary structure of the L subunit has been determined from the nucleotide sequence of the gene and from analyses of the amino and carboxyl termini of the protein. The sequences of a number of tryptic and chymotryptic peptides were used to corroborate the nucleotide sequence. The L subunit was found to be composed of 281 amino acids (Mr 31,319) and to contain five hydrophobic segments. It is homologous to the M subunit and to a plant thylakoid protein referred to as the QB or Mr 32,000 protein.     

Effect of specific mutations of tyrosine-(M)210 on the primary photosynthetic electron-transfer process in Rhodobacter sphaeroides.

We have measured the rate of the initial electron-transfer process as a function of temperature in reaction centers in a native strain of the photosynthetic bacterium Rhodobacter sphaeroides and two mutants generated by site-directed mutagenesis. In the mutants, a tyrosine residue in the vicinity of the primary electron donor and acceptor molecules was replaced by either phenylalanine or isoleucine. The electron-transfer reaction is slower in the mutants and has a qualitatively different dependence on temperature. In native reaction centers the rate increases as the temperature is reduced, in the phenylalanine mutant it is virtually independent of temperature, and in the isoleucine mutant it decreases with decreasing temperature. At 77 K, the electron-transfer reaction is approximately 30 times slower in the isoleucine mutant than in the native. These observations support the view that tyrosine-(M)210 plays an important role in the electron-transfer mechanism. In the isoleucine mutant at low temperatures, the stimulated emission from the excited reaction center undergoes a time-dependent shift to shorter wavelengths.     

Proton transfer from the bulk to the bound ubiquinone Q(B) of the reaction center in chromatophores of Rhodobacter sphaeroides: retarded conveyance by neutral water

The mechanism of proton transfer from the bulk into the membrane protein interior was studied. The light-induced reduction of a bound ubiquinone molecule Q(B) by the photosynthetic reaction center is accompanied by proton trapping. We used kinetic spectroscopy to measure (i) the electron transfer to Q(B) (at 450 nm), (ii) the electrogenic proton delivery from the surface to the Q(B) site (by electrochromic carotenoid response at 524 nm), and (iii) the disappearance of protons from the bulk solution (by pH indicators). The electron transfer to Q(B)(-) and the proton-related electrogenesis proceeded with the same time constant of approximately 100 microseconds (at pH 6.2), whereas the alkalinization in the bulk was distinctly delayed (tau approximately 400 microseconds). We investigated the latter reaction as a function of the pH indicator concentration, the added pH buffers, and the temperature. The results led us to the following conclusions: (i) proton transfer from the surface-located acidic groups into the Q(B) site followed the reduction of Q(B) without measurable delay; (ii) the reprotonation of these surface groups by pH indicators and hydronium ions was impeded, supposedly, because of their slow diffusion in the surface water layer; and (iii) as a result, the protons were slowly donated by neutral water to refill the proton vacancies at the surface. It is conceivable that the same mechanism accounts for the delayed relaxation of the surface pH changes into the bulk observed previously with bacteriorhodopsin membranes and thylakoids. Concerning the coupling between proton pumps in bioenergetic membranes, our results imply a tendency for the transient confinement of protons at the membrane surface.     

Lipopolysaccharide from Rhodobacter sphaeroides is an agonist in equine cells

Endotoxemia is associated with the principal causes of death in adult horses and equine neonates and, therefore, veterinary researchers are expending efforts to identify new therapeutic interventions that might be beneficial in these animals. Endotoxin antagonists inhibit interaction of endotoxin with cellular receptors and may be beneficial in the treatment of endotoxemia and sepsis. Diphosphoryl lipid A from Rhodobacter sphaeroides (RsDPLA) is a potent antagonist of enteric LPS in human cells, but is an agonist in hamster cells. In this study, the effect of lipopolysaccharide from R. sphaeroides (RsLPS) on equine whole blood and isolated monocyte preparations was investigated by comparing tumor necrosis factor (TNF) production in response to RsLPS and Escherichia coli O55:B5 LPS. Our results indicate that RsLPS is a potent agonist in equine cells, which precludes therapeutic use of this agent in equine patients. In contrast to the results in equine cells, RsLPS did not elicit TNF production by itself, and inhibited the response to E. coli O55:B5 LPS in a human monocytic cell line.