Structure of the membrane-bound protein photosynthetic reaction center from Rhodobacter sphaeroides

The structure of the photosynthetic reaction center (RC) from Rhodobacter sphaeroides was determined at 3.1-A resolution by the molecular replacement method, using the Rhodopseudomonas viridis RC as the search structure. Atomic coordinates were refined with the difference Fourier method and restrained least-squares refinement techniques to a current R factor of 22%. The tertiary structure of the RC complex is stabilized by hydrophobic interactions between the L and M chains, by interactions of the pigments with each other and with the L and M chains, by residues from the L and M chains that coordinate to the Fe2+, by salt bridges that are formed between the L and M chains and the H chain, and possibly by electrostatic forces between the ends of helices. The conserved residues at the N-termini of the L and M chains were identified as recognition sites for the H chain.     

Crystallization of the reaction center from Rhodobacter sphaeroides in a new tetragonal form

The reaction center from the nonsulfur purple bacteriumRhodobacter sphaeroideshas been crystallized in a new form. The crystals grew in the presence of polyethylene glycol 4000, the detergent β-octyl glucoside, and the amphiphiles heptane triol and benzamidine hydrochloride, using the sitting drop method. The space group of these crystals is tetragonal, P4₁(4₃)2₁2, and the cell constants are a = b = 141.5 Å and c = 276.7 Å with probably 2 proteins per asymmetric unit. A native data set has been set collected to a resolution of 2.8 Å consisting of 56,332 unique reflections (50,731 with F > 2σ) with anR_sym of 9.5%. Analysis of the diffraction data is underway using molecular and isomorphous replacement. © 1994 Wiley-Liss, Inc.     

Pigment-protein interactions in Rhodobacter sphaeroides Y photochemical reaction center; comparison with other reaction center structures

Structural characteristics of pigments and cofactors are analyzed in the X-ray structure of the Rhodobacter sphaeroides (Y strain) photochemical reaction center, recently refined at 3 Å resolution (Arnoux B, Gaucher JF, Ducruix A and Reiss-Husson F (1995) Acta Cryst D51: 368–379). As several structures are now available for these pigment-protein complexes from various Rhodobacter sphaeroides strains and for Rhodopseudomonas viridis, a detailed comparison was done for highlighting converging structural results as well as for pointing to incidental differences. Comparison of mean plane orientations and distances, and also direct superposition of the pigment arrays, indicated that the best agreement between all the structures concerned the dimer and the bacteriopheophytin of the A branch. In the Y reaction center structure the pentacoordination of the Mg⁺⁺ atoms of the bacteriochlorophylls, and the H bonding pattern of the porphyrin conjugated carbonyls are consistent with the better resolved Rhodobacter sphaeroides recently published structure (Ermler U, Fritzsch G, Buchanan SK and Michel H (1995) Structure 2:925–936). Discrepancies between the various Rhodobacter sphaeroides structures are larger for the quinones, particularly the secondary one. In the Y reaction center structure the phytyl and isoprenoid chains of the cofactors are defined and their local mobility was evaluated by analyzing the temperature factor and the density of neighbouring atoms. Significant differences were observed between the A and B branches, and, within each branch, from the dimer to the quinone molecules. Correspondence to: F. Reiss-Husson     

Photochemical trapping of a bacteriopheophytin anion in site-specific reaction-center mutants from the photosynthetic bacterium Rhodobacter sphaeroides.

The mutant YY in the reaction center of Rhodobacter sphaeroides, in which Phe181 on the L chain has been replaced by Tyr, and the double mutant FY, with Tyr210 on the M chain replaced by Phe and Phe181 on the L chain replaced by Tyr, have been constructed by site-directed mutagenesis. The studies described here were performed to complement a previous mutational analysis of mutant FF with Tyr210 replaced by Phe. Both new strains grow photoheterotrophically. The optical absorption spectra of reaction centers isolated from these mutants have band shifts attributable to the monomer bacteriochlorophylls in the vicinity of the substitutions. Photochemical trapping of the bacteriopheophytin anion (I-) indicates that the bacteriopheophytin on the B branch is reduced to a much greater extent in FF and FY as compared to YY and wild-type YF. Low temperature (77 K) absorption spectra clearly show that in the wild-type (YF) and YY reaction centers only the 545-nm-absorbing bacteriopheophytin is reduced while in the FF and FY reaction centers both the 535-nm and 545-nm-absorbing bacteriopheophytins are reduced. A simple kinetic analysis is used to explain these results. This analysis suggests that, in order for the observed trapping results to occur, a decrease in the 'cycling' time must take place, that is changes in the rate(s) of charge recombination must accompany the already known decrease in the forward electron transfer rate.     

Effect of high pressure on the photochemical reaction center from Rhodobacter sphaeroides R26.1

High-pressure studies on the photochemical reaction center from the photosynthetic bacterium Rhodobacter sphaeroides, strain R26.1, shows that, up to 0.6 GPa, this carotenoid-less membrane protein does not loose its three-dimensional structure at room temperature. However, as evidenced by Fourier-transform preresonance Raman and electronic absorption spectra, between the atmospheric pressure and 0.2 GPa, the structure of the bacterial reaction center experiences a number of local reorganizations in the binding site of the primary electron donor. Above that value, the apparent compressibility of this membrane protein is inhomogeneous, being most noticeable in proximity to the bacteriopheophytin molecules. In this elevated pressure range, no more structural reorganization of the primary electron donor binding site can be observed. However, its electronic structure becomes dramatically perturbed, and the oscillator strength of its Q(y) electronic transition drops by nearly one order of magnitude. This effect is likely due to very small, pressure-induced changes in its dimeric structure.     

Kinetic analysis of the thermal stability of the photosynthetic reaction center from Rhodobacter sphaeroides

The temperature-induced denaturation of the photosynthetic reaction center from Rhodobacter sphaeroides has been studied through the changes that occur in the absorption spectrum of the bound chromophores on heating. At elevated temperatures, the characteristic absorbance bands of the bacteriochlorins bound to the polypeptides within the reaction center are lost, and are replaced by features typical of unbound bacteriochlorophyll and bacteriopheophytin. The kinetics of the spectral changes cannot be explained by a direct conversion from the functional to the denatured form of the protein, and require the presence of at least one intermediate. Possible mechanisms for the transformation via an intermediate are examined using a global analysis of the kinetic data, and the most likely mechanism is shown to involve a reversible transformation between the native state and an off-pathway intermediate, coupled to an irreversible transformation to the denatured state. The activation energies for the transformations between the three components are calculated from the effect of temperature on the individual rate constants, and the likely structural changes of the protein during the temperature-induced transformation are discussed.     

DNA repair mutants of Rhodobacter sphaeroides.

The genome of the photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1 comprises two chromosomes and five endogenous plasmids and has a 65% G+C base composition. Because of these characteristics of genome architecture, as well as the physiological advantages that allow this organism to live in sunlight when in an anaerobic environment, the sensitivity of R. sphaeroides to UV radiation was compared with that of the more extensively studied bacterium Escherichia coli. R. sphaeroides was found to be more resistant, being killed at about 60% of the rate of E. coli. To begin to analyze the basis for this increased resistance, a derivative of R. sphaeroides, strain 2.4.1 delta S, which lacks the 42-kb plasmid, was mutagenized with a derivative of Tn5, and the transposon insertion mutants were screened for increased UV sensitivity (UVs). Eight UVs strains were isolated, and the insertion sites were determined by contour-clamped homogeneous electric field pulsed-field gel electrophoresis. These mapped to at least five different locations in chromosome I. Preliminary analysis suggested that these mutants were deficient in the repair of DNA damage. This was confirmed for three loci by DNA sequence analysis, which showed the insertions to be within genes homologous to uvrA, uvrB, and uvrC, the subunits of the nuclease responsible for excising UV damage.     

Effects of photosynthetic reaction center H protein domain mutations on photosynthetic properties and reaction center assembly in Rhodobacter sphaeroides

Purple bacterial photosynthetic reaction center (RC) H proteins comprise three cellular domains: an 11 amino acid N-terminal sequence on the periplasmic side of the inner membrane; a single transmembrane alpha-helix; and a large C-terminal, globular cytoplasmic domain. We studied the roles of these domains in Rhodobacter sphaeroides RC function and assembly, using a mutagenesis approach that included domain swapping with Blastochloris viridis RC H segments and a periplasmic domain deletion. All mutations that affected photosynthesis reduced the amount of the RC complex. The RC H periplasmic domain is shown to be involved in the accumulation of the RC H protein in the cell membrane, while the transmembrane domain has an additional role in RC complex assembly, perhaps through interactions with RC M. The RC H cytoplasmic domain also functions in RC complex assembly. There is a correlation between the amounts of membrane-associated RC H and RC L, whereas RC M is found in the cell membrane independently of RC H and RC L. Furthermore, substantial amounts of RC M and RC L are found in the soluble fraction of cells only when RC H is present in the membrane. We suggest that RC M provides a nucleus for RC complex assembly, and that a RC H/M/L assemblage results in a cytoplasmic pool of soluble RC M and RC L proteins to provide precursors for maximal production of the RC complex.     

Intracytoplasmic membrane vesiculation in light-harvesting mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Abstract In Chlamydomonas reinhardtii there are three glutamate dehydrogenase isozymes which can use both NADH and NADPH as cofactors and respond differently to different nitrogen sources and several stress conditions. From data of induction of isozymes in different metabolic situations, we propose a possible physiological role for each of them in algal carbon and nitrogen metabolism.     

Photosynthetic reaction center mutagenesis via chimeric rescue of a non-functional Rhodobacter capsulatus puf operon with sequences from Rhodobacter sphaeroides

Photosynthetically active chimeric reaction centers which utilize genetic information from both Rhodobacter capsulatus and Rb. sphaeroides puf operons were isolated using a novel method termed chimeric rescue. This method involves in vivo recombination repair of a Rb. capsulatus host operon harboring a deletion in pufM with a non-expressed Rb. sphaeroides donor puf operon. Following photosynthetic selection, three revertant classes were recovered: 1) those which used Rb. sphaeroides donor sequence to repair the Rb. capsulatus host operon without modification of Rb. sphaeroides puf operon sequences (conversions), 2) those which exchanged sequence between the two operons (inversions), and 3) those which modified plasmid or genomic sequences allowing expression of the Rb. sphaeroides donor operon. The distribution of recombination events across the Rb. capsulatus puf operon was decidedly non-random and could be the result of the intrinsic recombination systems or could be a reflection of some species-specific, functionally distinct characteristic(s). The minimum region required for chimeric rescue is the D-helix and half of the D/E-interhelix of M. When puf operon sequences 3 of nucleotide M882 are exchanged, significant impairment of excitation trapping is observed. This region includes both the 3 end of pufM and sequences past the end of pufM.     

Effect of chemical chaperones on properties of lysozyme and the reaction center protein from Rhodobacter sphaeroides

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and the functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. Selected chemical chaperones are strongly different by the structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described within the frames of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic nuclus) moieties. This fact provides additional regulation of functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in denaturation temperature T(d), and an increase in the amplitude of equilibrium fluctuation, which allows him to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in the case of methylresorcinol. Therefore, methylresorcinol does not effect the functional properties of the RC protein. It was concluded that various chaperones at one and the same concentration and chaperones at different concentrations form diverse 3D structures of proteins, which differ by dynamic and functional characteristics.     

Preferential binding of equine ferricytochrome c to the bacterial photosynthetic reaction center from Rhodobacter sphaeroides

Redox titration of horse heart cytochrome c (cyt c), in the presence of varying concentrations of detergent-solubilized photosynthetic reaction center (RC) from Rhodobacter sphaeroides, revealed an RC concentration-dependent decrease in the measured cyt c midpoint potential that is indicative of a 3.6 +/- 0.2-fold stronger binding affinity of oxidized cytochrome to a single binding site. This effect was correlated with preferential binding in the functional complex by redox titration of the fraction of RCs exhibiting microsecond, first-order, special pair reduction by cytochrome. A binding affinity ratio of 3.1 +/- 0.4 was determined by this second technique, confirming the result. Redox titration of flash-induced intracomplex electron transfer also showed the association in the electron transfer-active complex to be strong, with a dissociation constant of 0.17 +/- 0.03 microM. The tight binding is associated with a slow off-rate which, in the case of the oxidized form, can influence the kinetics of P(+) reduction. The pitfalls of the common use of xenon flashlamps to photoexcite fast electron-transfer reactions are discussed with relation to the first electron transfer from primary to secondary RC quinone acceptors. The results shed some light on the diversity of kinetic behavior reported for the cytochrome to RC electron-transfer reaction.     

Transient optical spectroscopy of single crystals of the reaction center from Rhodobacter sphaeroides wild-type 2.4.1

The photoactivity of the crystallized reaction centers from Rhodobacter sphaeroides wild-type strain 2.4.1 has been examined by light-induced absorption spectral changes associated with charge separation and triplet state formation in the reaction center. Upon excitation of a crystal at ambient redox potential, the primary donor 865 nm band bleaches reversibly. The kinetics of its recovery were found to be biphasic with rate constants 11.5 +/- 1.3 s-1 and 0.9 +/- 0.4 s-1 which correspond to lifetimes of 87.0 +/- 9.0 ms and 1.0 +/- 0.7 s, respectively. The ratio of the fast-to-slow component preexponential terms was 3.5 +/- 1.1 suggesting that the majority (78.9 +/- 13.0%) of the reaction centers in the crystals lack the secondary quinone, QB. The addition of sodium ascorbate to the crystals attenuates the 865 nm absorption change, and gives rise to strong carotenoid triplet-triplet absorption changes at 547 nm. These data indicate that the reaction center-bound carotenoid in the crystals is capable of accepting triplet energy from the primary donor triplet.     

Manganese oxidation by modified reaction centers from Rhodobacter sphaeroides

The transfer of an electron from exogenous manganese (II) ions to the bacteriochlorophyll dimer, P, of bacterial reaction centers was characterized for a series of mutants that have P/P(+) midpoint potentials ranging from 585 to 765 mV compared to 505 mV for wild type. Light-induced changes in optical and EPR spectra of the mutants were measured to monitor the disappearance of the oxidized dimer upon electron donation by manganese in the presence of bicarbonate. The extent of electron transfer was strongly dependent upon the P/P(+) midpoint potential. The midpoint potential of the Mn(2+)/Mn(3+) couple was calculated to decrease linearly from 751 to 623 mV as the pH was raised from 8 to 10, indicating the involvement of a proton. The electron donation had a second order rate constant of approximately 9 x 10(4) M(-1) s(-1), determined from the linear increase in rate for Mn(2+) concentrations up to 200 microM. Weak dissociation constants of 100-200 microM were found. Quantitative EPR analysis of the six-line free Mn(2+) signal revealed that up to seven manganese ions were associated with the reaction centers at a 1 mM concentration of manganese. The association and the electron transfer between manganese and the reaction centers could be inhibited by Ca(2+) and Na(+) ions. The ability of reaction centers with high potentials to oxidize manganese suggests that manganese oxidation could have preceded water oxidation in the evolutionary development of photosystem II.     

Energetics of proton transfer pathways in reaction centers from Rhodobacter sphaeroides. The Glu-H173 activated mutants

Electron transfer between the primary and secondary quinones (Q(A), Q(B)) in the bacterial photosynthetic reaction center (bRC) is coupled with proton uptake at Q(B). The protons are conducted from the cytoplasmic side, probably with the participation of two water channels. Mutations of titratable residues like Asp-L213 to Asn (inhibited mutant) or the double mutant Glu-L212 to Ala/Asp-L213 to Ala inhibit these electron transfer-coupled proton uptake events. The inhibition of the proton transfer (PT) process in the single mutant can be restored by a second mutation of Arg-M233 to Cys or Arg-H177 to His (revertant mutant). These revertant mutants shed light on the location of the main proton transfer pathway of wild type bRC. In contrast to the wild type and inhibited mutant bRC, the revertant mutant bRC showed notable proton uptake at Glu-H173 upon formation of the Q(B)- state. In all of these mutants, the pK(a) of Asp-M17 decreased by 1.4-2.4 units with respect to the wild type bRC, whereas a significant pK(a) upshift of up to 5.8 units was observed at Glu-H122, Asp-H170, Glu-H173, and Glu-H230 in the revertant mutants. These residues belonging to the main PT pathway are arranged along water channel P1 localized mainly in subunit H. bRC possesses subunit H, which has no counterpart in photosystem II. Thus, bRC may possess alternative PT pathways involving water channels in subunit H, which becomes active in case the main PT pathway is blocked.     

Crystallographic studies of mutant reaction centres from Rhodobacter sphaeroides

X-ray structures have been determined for five mutant reaction centres from Rhodobacter sphaeroides, at resolutions varying between 3.4 Å and 2.3 Å. The aim was to examine the effects of mutagenesis of polar residues in the binding pocket of the reaction centre carotenoid. The number of water molecules identified in each structure depended on the resolution and completeness of the data. In a 2.3 Å structure for a WM115F/FM197R mutant reaction centre, two water molecules partially filled the cavity that was created when the tryptophan residue was replaced by a less bulky phenylalanine. Structures obtained for four reaction centres with mutations of polar residues in the carotenoid binding pocket failed to show any significant change in the structure of the reaction centre carotenoid. Low resolution data for a YM210W mutant reaction centre showed that the overall structure of this complex is well conserved. This finding is discussed in light of the intriguing spectroscopic properties of the YM210W mutant reaction centre, and an alternative pathway for transmembrane electron transfer identified in this mutant.     

Energy trapping and detrapping by wild type and mutant reaction centers of purple non-sulfur bacteria

Time-correlated single photon counting was used to study energy trapping and detrapping kinetics at 295 K in Rhodobacter sphaeroides chromatophore membranes containing mutant reaction centers. The mutant reaction centers were expressed in a background strain of Rb. sphaeroides which contained only B880 antenna complexes and no B800-850 antenna complexes. The excited state decay times in the isolated reaction centers from these strains were previously shown to vary by roughly 15-fold, from 3.4 to 52 ps, due to differences in the charge separation rates in the different mutants (Allen and Williams (1995) J Bioenerg Biomembr 27: 275–283). In this study, measurements were also performed on wild type Rhodospirillum rubrum and Rb. sphaeroides B880 antenna-only mutant chromatophores for comparison. The emission kinetics in membranes containing mutant reaction centers was complex. The experimental data were analyzed in terms of a kinetic model that involved fast excitation migration between antenna complexes followed by reversible energy transfer to the reaction center and charge separation. Three emission time constants were identified by fitting the data to a sum of exponential decay components. They were assigned to trapping/quenching of antenna excitations by the reaction center, recombination of the P⁺H^– charge-separated state of the reaction center reforming an emitting state, and emission from uncoupled antenna pigment-protein complexes. The first varied from 60 to 160 ps, depending on the reaction center mutation; the second was 200–300 ps, and the third was about 700 ps. The observed weak linear dependence of the trapping time on the primary charge separation time, together with the known sub-picosecond exciton migration time within the antenna, supports the concept that it is energy transfer from the antenna to the reaction center, rather than charge separation, that limits the overall energy trapping time in wild type chromatophores. The component due to charge recombination reforming the excited state is minor in wild type membranes, but increases substantially in mutants due to the decreasing free energy gap between the states P^* and P⁺H^–.Abbreviations PSU photosynthetic unit - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - P reaction center primary electron donor - RC reaction center - Rb. Rhodobacter - Rs. Rhodospirillum - EDTA (ethylenediamine)tetraacetic acid - Tris tris(hydroxymethyl)aminomethane Author for correspondence     

Brominated lipids identify lipid binding sites on the surface of the reaction center from Rhodobacter sphaeroides

This study describes the use of brominated phospholipids to distinguish between lipid and detergent binding sites on the surface of a typical alpha-helical membrane protein. Reaction centers isolated from Rhodobacter sphaeroides were cocrystallized with added brominated phospholipids. X-ray structural analysis of these crystals has revealed the presence of two lipid binding sites from the characteristic strong X-ray scattering from the bromine atoms. These results demonstrate the usefulness of this approach to mapping lipid binding sites at the surface of membrane proteins.