Isolation of two new natural pteridines from photosynthetic bacteria, Rhodopseudomonas sphaeroides.

Two new natural pteridines have been isolated from the cultured medium of Rhodopseudomonas sphaeroides GM-1. The compounds are tentatively identified as 2-amino-4-hydroxy-6-hydroxy-6-(1,2, 3,4-tetrahydroxybutyl)pteridine and 2-amino-4-hydroxy-6-(3-hydroxy-4-phosphonoxy-1-butenyl)pteridine by degradative experiments and by electrophoretic and paper chromatographic comparison with authentic materials.

Comparison of reaction centers from Rhodobacter sphaeroides and Rhodopseudomonas viridis: overall architecture and protein-pigment interactions.

Photosynthetic reaction centers (RCs) from the photosynthetic bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis are protein complexes closely related in both structure and function. The structure of the Rps. viridis RC was used to determine the structure of the RC from Rb. sphaeroides. Small but meaningful differences between the positions of the helices and the cofactors in the two complexes were identified. The distances between helices AL and AM, between BL and BM, and between bacteriopheophytins BPL and BPM are significantly shorter in Rps. viridis than they are in Rb. sphaeroides RCs. There are a number of differences in the amino acid residues that surround the cofactors; some of these residues form hydrogen bonds with the cofactors. Differences in chemical properties and location of these residues account in some manner for the different spectral properties of the two RCs. In several instances, the hydrogen bonds, as well as the apparent distances between the histidine ligands and the Mg atoms of the bacteriochlorophylls, were found to significantly differ from the Rb. sphaeroides RC structure previously described by Yeates et al. [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7993-7997] and Allen et al. [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8487-8491].

Sub-picosecond dynamics of excited state of primary electron donor in reaction centers of Rhodopseudomonas viridis as revealed by hole burning at 1.7K broad and narrow holes.

Within the QF band of the primary electron donor (P), the spectra of absorbance changes due to the formation of a state of P+QA- (QA is the primary quinone) at 1.7K in Rhodopseudomonas viridis reaction centers excited at 1014 nm have been shown to involve two spectral features characterized by: (i) a progression of broad (170-190 cm-1) Gaussian vibronic bands (S-factor = 1.4) separated by 150 cm-1 and (ii) a 'narrow' structure near 1014 nm, characterized by 0-0 transition at 1014 nm with a width of approximately 50 cm-1 and 0-1 transition at 1000 nm with the width of approximately 100 cm-1, and S-factor = 0.9. The width of 50 cm-1 can be related to either zero-phonon hole (ZPH) width or the structure involving phonon wings and ZPH being unresolved. Since dichroic value (approximately 0.37) is unvarying over the P band, the vibrations involved are totally symmetric. The ZPH (width of approximately 3 cm-1) and phonon wings (frequency of approximately 30 cm-1) are resolved within the P band near 1014 nm when the spectrum of delta A due to the formation of bacteriopheophytinL- is measured at 1.7K.

Partial symmetrization of the photosynthetic reaction center.

The bacterial photosynthetic reaction center (RC) is a pigmented intrinsic membrane protein that performs the primary charge separation event of photosynthesis, thereby converting light to chemical energy. The RC pigments are bound primarily by two homologous peptides, the L and M subunits, each containing five transmembrane helices. These alpha helices and pigments are arranged in an approximate C2 symmetry and form two possible electron transfer pathways. Only one of these pathways is actually used. In an attempt to identify nonhomologous residues that are responsible for functional differences between the two branches, homologous helical regions that interact extensively with the pigments were genetically symmetrized (that is, exchanged). For example, replacement of the fourth transmembrane helix (D helix) in the M subunit with the homologous helix from the L subunit yields photosynthetically inactive RCs lacking a critical photoactive pigment. Photosynthetic revertants have been isolated in which single amino acid substitutions (intragenic suppressors) compensate for this partial symmetrization.

Electrostatic control of charge separation in bacterial photosynthesis.

Electrostatic interaction energies of the electron carriers with their surroundings in a photosynthetic bacterial reaction center are calculated. The calculations are based on the detailed crystal structure of reaction centers from Rhodopseu-domonas viridis, and use an iterative, self-consistent procedure to evaluate the effects of induced dipoles in the protein and the surrounding membrane. To obtain the free energies of radical-pair states, the calculated electrostatic interaction energies are combined with the experimentally measured midpoint redox potentials of the electron carriers and of bacteriochlorophyll (BChl) and bacteriopheophytin (BPh) in vitro. The P+HL- radical-pair, in which an electron has moved from the primary electron donor (P) to a BPh on the 'L' side of the reaction center (HL), is found to lie approx. 2.0 kcal/mol below the lowest excited singlet state (P*), when the radical-pair is formed in the static crystallographic structure. The reorganization energy for the subsequent relaxation of P+HL- is calculated to be 5.0 kcal/mol, so that the relaxed radical-pair lies about 7 kcal/mol below P*. The unrelaxed P+BL- radical-pair, in which the electron acceptor is the accessory BChl located between P and HL, appears to be essentially isoenergetic with P*.P+BM-, in which an electron moves to the BChl on the 'M' side, is calculated to lie about 5.5 kcal/mol above P*. These results have an estimated error range of +/- 2.5 kcal/mol. They are shown to be relatively insensitive to various details of the model, including the charge distribution in P+, the atomic charges used for the amino acid residues, the boundaries of the structural region that is considered microscopically and the treatments of the histidyl ligands of P and of potentially ionizable amino acids. The calculated free energies are consistent with rapid electron transfer from P* to HL by way of BL, and with a much slower electron transfer to the pigments on the M side. Tyrosine M208 appears to play a particularly important role in lowering the energy of P+BL-. Electrostatic interactions with the protein favor localization of the positive charge of P+ on PM, one of the two BChl molecules that make up the electron donor.

Soluble cytochromes and ferredoxins from the marine purple phototrophic bacterium, Rhodopseudomonas marina.

Four soluble c-type cytochromes, the high redox potential 4-Fe-S ferredoxin known as HiPIP, a large molecular weight 2-Fe-S ferredoxin and a 4-Fe-S 'bacterial' ferredoxin, were isolated from extracts of two strains of Rps. marina. Cytochrome c-550, cytochrome c' and cytochrome c-549 were previously described, and we have extended their characterization. Cytochrome c-558, which has not previously been observed in Rps. marina, appears to be a low-spin isozyme of the more commonly observed high-spin cytochrome c'. HiPIP, which was not observed in previous work, was found to be abundant in Rps. marina. The 2-Fe-S ferredoxin, which has previously been observed only in Rps. palustris, has a native size greater than 100 kDa and a subunit size of 17 kDa. The 'bacterial' ferredoxin appears to have only a single four-iron-sulfur cluster. We examined photosynthetic membranes by difference spectroscopy and found abundant c-type cytochromes. Approximately one-quarter of the heme can be reduced by ascorbate and the remainder by dithionite. There is 2 nm difference between the high-potential heme (554 nm) and the low (552 nm). These characteristics resemble those of the tetraheme reaction center cytochrome of Rps. viridis. In addition to the electron transfer components, we found small amounts of a fluorescent yellow protein which has spectral resemblance to a photoactive yellow protein from Ec. halophila.

Structural analysis of the nontoxic lipid A of Rhodobacter capsulatus 37b4.

Lipid A from Rhodobacter capsulatus 37b4 consists of a D-glucosaminyl-(beta 1-6)-D-glucosamine disaccharide backbone, carrying diphosphorylethanolamine at C-1 of the reducing glucosamine and phosphorylethanolamine at C-4' of the nonreducing glucosamine. 1,4'-Bisphosphorylated lipid A, lacking the polar head groups, was also encountered and contributed to the observed microheterogeneity in the phosphate substitution. The amino functions of both glucosamines are substituted almost entirely by the rare 3-oxotetradecanoic acid, which is a characteristic constituent of lipid A in the genus Rhodobacter. 3-Hydroxydecanoic acid is ester-bound at C-3 and C-3' of the glucosamine disaccharide and the one at the nonreducing glucosamine (C-3') is partially substituted by dodecenoic acid to form an ester-bound diester. In free lipid A, hydroxy groups at C-4 and C-6' of the glucosamine disaccharide are unsubstituted. C-6' being the putative attachment point of the lipopolysaccharide core. The nontoxic Rhodobacter capsulatus lipid A shows extensive serological cross-reaction with the toxic Salmonella lipid A. Structural similarities in the hydrophilic part of both types of lipid A, dissimilarities in the hydrophobic part and their impacts on serologic properties are discussed.

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.

The hopanoids of the purple non-sulfur bacteria Rhodopseudomonas palustris and Rhodopseudomonas acidophila and the absolute configuration of bacteriohopanetetrol.

Five complex hopanoids have been detected in the purple non-sulfur bacterium Rhodopseudomonas acidophila. Next to the polyfunctionalized methylcyclopentane bacteriohopanetetrol ether already isolated from Methylobacterium organophilum, 35-carbamoylbacteriohopane-32,33,34-triol, 34,35-dicarbamoylbacteriohopane-32,33-diol and two nucleoside analogues, (22R)-30-(5'-adenosyl)hopane and (22S)-30-(5'-adenosyl)hopane were isolated and identified by spectroscopic and chemical methods. In Rhodopseudomonas palustris, however, only 35-amino-bacteriohopane-32,33,34-triol was detected. Chemical correlation between adenosylhopane and bacteriohopanetetrol, as well as comparison of derivatives obtained from bacterial and synthetic hopanoids, permitted the determination of the configurations of all asymmetric centres of the side-chain of bacteriohopanetetrol as 22R, 32R, 33R and 34S. According to the stereochemistry, this side-chain could be a D-ribose derivative linked through its C-5 carbon atom to the hopane skeleton.

The amino acid sequence of the cytochrome c2 from the phototrophic bacterium Rhodopseudomonas globiformis.

The amino acid sequence of the principal soluble cytochrome c from the phototrophic acidophilic bacterium Rhodopseudomonas (or Rhodopila) globiformis was determined. By the criteria of percentage sequence identity and fewness of internal insertions and deletions it is more similar in sequence to some mitochondrial cytochromes c than to any known bacterial cytochrome. The organism does not have any properties that commend it as being particularly similar to postulated prokaryotic precursors of the mitochondrion. We consider that the relatively high degree of sequence similarity is an instance of convergence, and is an example of the limitations that are imposed on attempts to deduce distant evolutionary relationships from sequence information. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50136 (12 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].

Crystallization and preliminary analysis of crystals of cytochrome c2 from Rhodopseudomonas capsulata.

Two crystal forms of the cytochrome c2 isolated from Rhodopseudomonas capsulata have been obtained. One crystal form (type I), grown from ammonium sulfate solutions at pH 7.5, belongs to the space group R32 with unit cell dimensions of a = b = 100.0 A, and c = 162.2 A in the hexagonal setting. These crystals most likely contain two molecules in the asymmetric unit. The other crystal form (type II) was obtained from polyethylene glycol 6000 solutions at pH 6.5. Type II crystals belong to the space group P3(1)21 or P3(2)21 with one molecule per asymmetric unit and unit cell dimensions of a = b = 52.4 A, and c = 87.9 A. Both crystal forms diffract to at least 1.8 A resolution and appear to be resistant to radiation damage.

Structural homology of reaction centers from Rhodopseudomonas sphaeroides and Rhodopseudomonas viridis as determined by x-ray diffraction.

Crystals of the reaction center (RC) from Rhodopseudomonas sphaeroides with the space group P2(1)2(1)2(1), have been studied by x-ray diffraction. The Patterson search (molecular replacement) technique was used to analyze the data, with the structure of the reaction center from Rhodopseudomonas viridis as a model system. A preliminary electron density map of the reaction center from R. sphaeroides has been obtained. Comparison of the structure of the RC from R. sphaeroides with that from R. viridis showed the following conserved features: five membrane-spanning helices in each of the L and M subunits, a single membrane-spanning helix in the H subunit, a 2-fold symmetry axis, and similar positions and orientations of the cofactors. Unlike the RCs from R. viridis, both quinones are retained in the RCs from R. sphaeroides. The secondary quinone is located near the position related by the 2-fold symmetry axis to the primary quinone.

Crystallization and preliminary X-ray diffraction study of ferrocytochrome c2 from Rhodopseudomonas viridis.

Crystals of ferrocytochrome c2 from a non-sulphur purple photosynthetic bacterium, Rhodopseudomonas viridis, have been grown from ammonium sulphate solution at pH 8.5 by the sitting-drop vapour-diffusion procedure. The crystals belong to the trigonal system, space group P3(1)21 (or its enantiomorph P3(2)21) with unit-cell dimensions of a = b = 75.8 A and c = 40.1 A, and diffract to at least 2.0 A resolution. Assuming that an asymmetric unit contains one protein molecule (approx. 12,300 Mr), the solvent content of the crystal is approximately 54.5% (v/v).

Localization of the exposed N-terminal region of the B800-850 alpha and beta light-harvesting polypeptides on the cytoplasmic surface of Rhodopseudomonas capsulata chromatophores.

Proteinase K and trypsin were used to determine the orientation of the light-harvesting B800-850 alpha and beta polypeptides within the chromatophores (inside-out membrane vesicles) of the mutant strain Y5 of Rhodopseudomonas capsulata. With proteinase K 7 amino acid residues of the B800-850 alpha polypeptide were cleaved off up to position Trp-7--Thr-8 of the N terminus, and 11 residues were cleaved off up to position Leu-11-Ser-12 of the beta chain N terminus. The C termini of the B800-850 alpha and beta polypeptides, including the hydrophobic transmembrane portions, remained intact. It is proposed that the N termini of the alpha and beta subunits, each containing one transmembrane alpha-helical span, are exposed on the cytoplasmic membrane surface and the C termini are exposed to or directed toward the periplasm.

Isolation, characterization, and comparison of a ubiquitous pigment-protein complex consisting of a reaction center and light-harvesting bacteriochlorophyll proteins present in purple photosynthetic bacteria.

Protein complexes (photochemical reaction complex; PR complex) bound to both light-harvesting bacteriochlorophyll-1 (LH-Bchl-1) and reaction center Bchl (RC-Bchl) were purified from Rhodospirillum rubrum (wild and carotenoid-less), Rhodopseudomonas sphaeroides (wild), and Chromatium vinosum (wild). Another protein complex (LH-2 complex) bound to LH-Bchl-2 was also purified from Rps. sphaeroides. The bacteria were grown in the presence of a [14C]amino acid mixture. The purification procedure included molecular-sieve chromatography in the presence of cholate-deoxycholate, and non-equilibrated isoelectric electrophoresis with 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate. The purified complexes were separated into their constituent proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The molar ratios of the proteins were determined by comparing their radioactivities divided by their molecular weights after consideration of the molecular masses of the complexes. The PR complexes all contained per mol: 1 mol each of RC H-, M-, and L-subunits, 10-13 (probably 12) mol each of two other proteins with molecular weights of 11-12K and 8-11K, 28-32 mol Bchl, 13-15 mol carotenoids (except in the carotenoid-less mutant), 2.6-3.9 mol ubiquinone (or menaquinone in Chr. vinosum), and 53-79 mol phosphate without phospholipid. The LH-2 complex contained per mol: 1 mol 52K protein, about 13 (probably 12) mol each of 9K and 8K proteins, 30 mol Bchl, 10 mol carotenoids, and 38 mol phosphate without phospholipid. The PR complexes and LH-2 complex showed similar X-ray diffraction patterns, implying that they had similar, highly organized molecular structures.

Reversible chemical cross-linking of the light-harvesting polypeptides of Rhodopseudomonas viridis.

The topography of the light-harvesting polypeptides of Rhodopseudomonas viridis was investigated using cleavable chemical cross-linkers. To this end a set of succinimidyl esters and surface-specific sulfosuccinimidyl esters of different span widths were synthesized. The cross-linking reagents have been characterized using NMR and infrared spectroscopy and thin-layer chromatography. The cross-linking reaction was carried out under physiological conditions and the aggregates were analyzed by the methods of one- and two-dimensional polyacrylamide gel electrophoresis and by immunoblot analysis. We found cross-linkage between B1015-alpha and B1015-alpha, between B1015-alpha and B1015-beta and B1015-beta and B1015-beta. Aggregates of higher molecular mass were hetero-oligomers of B1015-alpha and B1015-beta containing three and four polypeptides, respectively. The results obtained in this work indicate a very tight contact among the light-harvesting polypeptides. We assume that the light-harvesting polypeptides are localized alternately as dimers of B1015-alpha and B1015-beta around the reaction centre core.

A novel hopanoid, 30-(5'-adenosyl)hopane, from the purple non-sulphur bacterium Rhodopseudomonas acidophila, with possible DNA interactions.

A novel hopanoid, 30-(5'-adenosyl)hopane, was isolated from the purple non-sulphur bacterium Rhodopseudomonas acidophila and identified. The significance of this triterpenoid in terms of bacteriohopanepolyol biosynthesis, membrane reinforcement and possible interactions with nucleic acids is discussed.

Pigment-protein complexes from Rhodopseudomonas palustris: isolation, characterization, and reconstitution into liposomes.

We have employed detergent solubilization and sucrose density gradient centrifugation to obtain pigment-protein complexes from Rhodopseudomonas palustris. Two types of detergent buffers were used, containing either octyl-beta-glucopyranoside (OG) plus sodium dodecyl sulfate (SDS) or OG alone. The fractions thus obtained were analyzed spectrophotometrically and by polyacrylamide gel electrophoresis to determine their pigment and protein composition. OG-SDS solubilization yields four fractions. The least dense of these fractions (OG-SDS a and b) are nonspecific mixtures of peptides and pigments. The next fraction, OG-SDS c, is an accessory light-harvesting complex, LHII, called B800-850. The largest particle, OG-SDS d, is a combination of reaction center (RC) and primary light-harvesting complex (LHI), B880. Solubilization using OG alone yields one fraction, a single large complex consisting of RC, LHI, and LHII. We have inserted the two large OG-SDS complexes and the OG complex into phospholipid liposomes to determine the size of such complexes in freeze-fractured membranes. On the basis of morphological, biochemical, and available biophysical data, we propose the following models for pigment-protein complexes in R. palustris membranes: 5-nm particles as free RC or LHI tetramers, 7.5-nm particles as LHI or LHII octamers (or both); 10-nm particles as RC-LHI core complexes (1 RC plus 12 LHI) or large LHII oligomers (or both), and large particles of 12.5 and 15 nm and LHII associated with the RC-LHI core complex.

X-ray structure analysis of a membrane protein complex. Electron density map at 3 A resolution and a model of the chromophores of the photosynthetic reaction center from Rhodopseudomonas viridis.

X-ray analysis of three-dimensional crystals of the photosynthetic reaction center from the purple bacterium Rhodopseudomonas viridis led to an electron density distribution at 3 A resolution calculated with phases from multiple isomorphous replacement. The protein subunits of the complex were identified. An atomic model of the prosthetic groups of the reaction center complex (4 bacteriochlorophyll b, 2 bacteriopheophytin b. 1 non-heme iron, 1 menaquinone, 4 heme groups) was built. The arrangement of the ring systems of the bacteriochlorophyll b and bacteriopheophytin b molecules shows a local 2-fold rotation symmetry; two bacteriochlorophyll b form a closely associated, non-covalently linked dimer ("special pair"). A different local 2-fold symmetry axis is observed for the heme groups of the cytochrome part.