The light-harvesting polypeptides of Rhodospirillum rubrum. I. The amino-acid sequence of the second light-harvestng polypeptide B 880-beta (B 870-beta) of Rhodospirillum rubrum S 1 and the carotenoidless mutant G-9+. carotenoidless mutant G-9+

The light-harvesting complex B 880 from Rhodospirillum rubrum S 1 (wild type) and B 870 from the carotenoidless mutant G-9+ was shown to consist mainly of an organic solvent-(chloroform/methanol-) soluble and an organic solvent-insoluble polypeptide. The isolation and separation of these two low-molecular-mass polypeptides (Mr 6101 and Mr 6079) were achieved by a two-step extraction procedure of chromatophores using in the first step chloroform/methanol containing 0.1M ammonium acetate. Following Sephadex LH-60 chromatography of this first extract a light-harvesting polypeptide (B 870-alpha) was isolated and its complete amino acid sequence was determined (R. Brunisholz et al. (1981) FEBS Lett. 129/1, 150-154, B 880-alpha: G. Gogel et al. (1983) Biochim. Biophys. Acta 746, 32-39). Upon reextraction of the chromatophore pellet with chloroform/methanol/ammonium acetate containing in addition acetic acid a second low-molecular-mass polypeptide (B 880-beta of B 870-beta) was generated. The complete amino acid sequences of the chloroform/methanol-insoluble light-harvesting polypeptide of Rs. rubrum S 1 (B 880-beta) and of Rs. rubrum G-9+ (B 870-beta) were determined. They are identical and consist of 54 amino acid residues. The conserved histidine residue within the hydrophobic stretch raises more evidence for ligand complexation of bacteriochlorophyll to this specific histidine residue which therefore possibly plays the key role in pigment-protein interactions. Both polypeptides (B 880-alpha and B 880-beta) are part of the light-harvesting complex B 880 in an apparent ratio of 1:1. Based on the primary structure data a possible arrangement of both light-harvesting polypeptides within the membrane will be discussed.     

Membrane topology of light-harvesting protein B870-alpha of Rhodospirillum rubrum G-9+. Amino acid residues in contact with the lipid bilayer as inferred from labeling with photogenerated carbenes

The amino acid residues of the light-harvesting protein B870-alpha of Rhodospirillum rubrum G-9+ that in the chromatophore membranes are in contact with the lipid phase were identified by hydrophobic photolabeling. Three reagents have been used which all contained the trifluoromethyldiazirinylphenyl group as a photo-sensitive precursor of a carbene but which otherwise differed in shape, molecular structure, and in the way they interact with membranes. 3-Trifluoromethyl-3-(m-[125I]iodophenyl)diazirine is a highly lipid-soluble compound, 11-[4-[(trifluoromethyl)diazirinyl]-phenyl]-[10-3H] 9-oxaundecanoic acid is an analogue of a fatty acid, and 1-palmitoyl-2-[11-[(trifluoromethyl)diaziri-nyl] phenyl]-[10-3H]9-oxaundecanoyl]-sn-glycero-3-phosphorylcholine one of a lecithin. Following labeling of chromatophores with these reagents, B870-alpha was isolated and subjected to (solid phase) Edman degradations in order to determine individual amino acid residues labeled. The main features of these results are as follows. 1) Labeling occurred both within the N-terminal segment (residues 1-8) and within the predominantly hydrophobic transmembrane stretch (residues 14-33). 2) Label distribution patterns within segments are indicative of helical structures to which the reagents had access to one face only of the cylindrical envelopes. 3) With regard to the transmembrane segment, the label distribution patterns were similar for all reagents whereas striking differences were noticed within the N-terminal portion. The labeling patterns are consistent with previous models proposing tight association of the transmembrane helix with that of the B870-beta chain. They also suggest that the N-terminal segment forms an amphipathic helix which interacts with the water-lipid interface of the membrane.     

Localisation of the subunits of the photosynthetic reaction centers in the chromatophore membrane of Rhodospirillum rubrum.

Reaction centers were isolated with the detergent lauryl dimethyl amine oxide from chromatophore membranes of Rhodospirillum rubrum. The subunit composition of these reaction centers is similar to the one obtained from Rhodopseudomonas spheroides: three subunits with the molecular weights of 21 000, 24 000 and 29 000. Reaction centers prepared from chromatophores labeled with 131I were heavely labeled in their large subunit (H). The smaller subunits (L and M) contained only little label. Sonication during labeling yielded a slightly higher incorporation of 131I in subunit H compared to the smaller ones. It is concluded that the H protein is largely exposed at the cytoplasmic side of the membrane but might also be accessible for iodination on the inside of the membrane while the L and M proteins are almost completely embedded in the membrane. Iodination of spheroplasts results in only a slight binding of 131I to chromatophores and reaction centers.     

The light-harvesting polypeptides of Rhodopseudomonas viridis. The complete amino-acid sequences of B1015-alpha, B1015-beta and B1015-gamma

Three low molecular mass polypeptides have been isolated by using the technique of organic solvent extraction of thylakoid membranes or whole cells from Rhodopseudomonas viridis. Their primary structures were determined by long liquid phase sequencer runs, combined with the isolation and sequence analysis of the C-terminal o-iodosobenzoic acid fragment and carboxypeptidase degradation. The polypeptide which consists of 58 amino-acids and is 46% homologous to the antenna polypeptide B880-alpha from Rhodospirillum rubrum was designated as B1015-alpha (1 His residue). The sequence homology between the second polypeptide, named B1015-beta (55 amino acids, 2 His residues) and B880-beta from Rs. rubrum is 52%. For the third polypeptide consisting of 36 amino acids and exhibiting a high hydrophobicity, no equivalent polypeptide has so far been found in other purple bacteria. The molar ratio of these three organic solvent soluble polypeptides from Rp. viridis was estimated to be 1:1:1. Accordingly, the 36 amino-acid polypeptide is likely to be an additional constituent of the light-harvesting complex B1015, consequently termed as B1015-gamma. According to hydrophathy profiles, the transmembrane arrangement of B1015-alpha and B1015-beta within the thylakoid membrane is supposed to be similar. B1015-gamma, however, shows a somewhat different hydropathy profile. A particular feature of this polypeptide is its high amount of aromatic amino acids. It is postulated that B1015-gamma is involved in the formation of regular arrays of light-harvesting complexes.     

Uridylylation of the P(II) protein in the photosynthetic bacterium Rhodospirillum rubrum.

The regulatory protein P(II) has been studied in great detail in enteric bacteria; however, its function in photosynthetic bacteria has not been clearly established. As a number of these bacteria have been shown to regulate nitrogenase activity by a metabolic control system, it is of special interest to establish the role of P(II) in these diazotrophs. In this study, we show that P(II) in Rhodospirillum rubrum is modified in response to the N status in the cell and that addition of ammonium or glutamine leads to demodification. We also provide evidence that P(II) is uridylylated. In addition, we show that not only these compounds but also NAD+ promotes demodification of P(II), which is of particular interest as this pyridine nucleotide has been shown to act as a switch-off effector of nitrogenase. Demodification of P(II) by ammonium or NAD+ did not occur in cultures treated with an inhibitor of glutamine synthetase (methionine sulfoximine), whereas treatment with the glutamate synthase inhibitor 6-diazo-5-oxo-norleucine led to total demodification of P(II) without any other addition. The results indicate that P(II) probably is not directly involved in darkness switch-off of nitrogenase but that a role in ammonium switch-off cannot be excluded.     

Methionine oxidation and its effect on the stability of a reconstituted subunit of the light-harvesting complex from Rhodospirillum rubrum.

An additional component in the purified core light-harvesting complex (LH1) from wild-type purple photosynthetic bacterium Rhodospirillum rubrum has been identified as an oxidized species of alpha-polypeptide by MALDI-TOF mass spectrometry. This component appears as a slightly earlier-eluting peak in the RP-HPLC chromatogram compared with the authentic alpha-polypeptide. The oxidation site has been determined to be the N-terminal methionine residue by high-resolution NMR spectroscopy, where the methionine is oxidized to methionine sulfoxide in a diastereoisomeric form. Interconversion between the oxidized and authentic alpha-polypeptides has been confirmed by selective oxidation and reduction. The oxidative modification of methionine is shown to have discernible effects on the ability to form B820 subunit with beta-polypeptide and bacteriochlorophyll a, and on the stability of the reconstituted B820 subunit. Both the ability and the stability for the samples using the oxidized alpha-polypeptide are moderately reduced, indicating that the oxidation-induced conformational change in the N-terminal domain of alpha-polypeptide may affect the pigment-binding environment through a long-range interaction. The MALDI-TOF mass results also reveal that the N-terminus of alpha-polypeptide is formylated and no phosphorylation has occurred in this polypeptide.     

Energy migration in the light-harvesting antenna of the photosynthetic bacterium Rhodospirillum rubrum studied by time-resolved excitation annihilation at 77 K.

The intensity dependence of picosecond kinetics in the light-harvesting antenna of the photosynthetic bacterium Rhodospirillum rubrum is studied at 77 K. By changing either the average excitation intensity or the pulse intensity we have been able to discriminate singlet-singlet and singlet-triplet annihilation. It is shown that the kinetics of both annihilation types are well characterized by the concept of percolative excitation dynamics leading to the time-dependent annihilation rates. The time dependence of these two types of annihilation rates is qualitatively different, whereas the dependencies can be related through the same adjustable parameter-a spectral dimension of fractal-like structures. The theoretical dependencies give a good fit to the experimental kinetics if the spectral dimension is equal to 1.5 and the overall singlet-singlet annihilation rate is close to the value obtained at room temperature. The percolative transfer is a consequence of spectral inhomogeneous broadening. The effect is more pronounced at lower temperatures because of the narrowing of homogeneous spectra.     

Organization of the genes coding for the reaction-centre L and M subunits and B870 antenna polypeptides α and β from the aerobic photosynthetic bacterium Erythrobacter species OCH114

In the aerobic photosynthetic bacterium Erythrobacter species OCH114 the structural genes coding for the light-harvesting (LH) complex B870 and the reaction-centre (RC) polypeptides (the gene products of the pufB, pufA, pufL and pufM genes) are mapped on a 2.728 kbp EcoRI fragment. Sequencing of this fragment revealed that the deduced amino acid sequences contain 50 (B870 beta), 52 (B850 alpha), 283 (RCL) and 331 (RCM) residues with the corresponding molecular weights of 5592, 5814, 31364, and 37671, respectively. In the corresponding mRNA a 'hairpin' structure (delta G degrees = -26.6 kcal) is predicted to be located immediately downstream of pufA. The RC and LH polypeptides are highly homologous to those of the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis. Directly downstream of pufM there is an open reading frame (ORF) of unknown size. Partial sequencing indicates that this ORF is highly homologous to the cytochrome subunit of the photosynthetic reaction centre from R. viridis. In the puf operon no pufQ or pufX genes could be found, but the bchA gene is located upstream of that operon. Plasmid pESS8.9 containing the 2.728 kbp EcoRI fragment reconstituted a photoinactive mutant of Erythrobacter species OCH114. Comparative analysis of the DNA region upstream of the puf operon and of bacteriochlorophyll (Bchl) synthesis indicated that Bchl synthesis and puf gene expression are regulated differently in Erythrobacter and purple bacteria, respectively.     

Transverse membrane topography of the B875 light-harvesting polypeptides of wild-type Rhodobacter sphaeroides.

Purified B875 light-harvesting complex, chromatophores, and spheroplast-derived vesicles from wild-type Rhodobacter sphaeroides were treated with proteinase K or trypsin, and the alpha and beta polypeptides were analyzed by electrophoretic, immunochemical, and protein-sequencing methods. With the purified complex, proteinase K digested both polypeptides and completely eliminated the A875 peak. Trypsin digested the alpha polypeptide and reduced the A875 by 50%. Proteinase K cleaved the beta polypeptide of chromatophores and the alpha polypeptide of spheroplast-derived vesicles. Sequence analyses of polypeptides extracted from proteinase K-treated chromatophores revealed that the beta polypeptide was cleaved between amino acids 4 and 5 from the N terminus. The N terminus of the alpha polypeptide was intact. We concluded that the N terminus of the beta polypeptide is exposed on the cytoplasmic membrane surface, and the difference in the digestion patterns between the spheroplast-derived vesicles and chromatophores suggested that the C terminus of the alpha polypeptide is exposed on the periplasmic surface.     

The light-harvesting chlorpohyll-protein complex of photosystem II. Its location in the photosynthetic membrane

We have investigated the structure of the photosynthetic membrane in a mutant of barley known to lack a chlorophyll-binding protein. This protein is thought to channel excitation energy to photosystem II, and is known as the "light-harvesting chlorophyll-protein complex." Extensive stacking of thylakoids into grana occurs in both mutant and wild-type chloroplasts. Examination of membrane internal structure by freeze-fracturing indicates that only slight differences exist between the fracture faces of mutant and wild-type membranes. These differences are slight reductions in the size of particles visible on the EFs fracture face, and in the number of particles seen on the PFs fracture face. No differences can be detected between mutant and wild-type on the etched out surface of the membrane. In contrast, tetrameric particles visible on the etched inner surface of wild-type thylakoids are extremely difficult to recognize on similar surfaces of the mutant. These particles can be recognized on inner surfaces of the mutant membranes when they are organized into regular lattices, but these lattices show a much closer particle-to-particle spacing than similar lattices in wild-type membranes. Although several interpretations of these data are possible, these observations are consistent with the proposal that the light-harvesting chlorophyll-protein complex of photosystem II is bound to the tetramer (which is visible on the EFs face as a single particle) near the inner surface of the membrane. The large tetramer, which other studies have shown to span the thylakoid membrane, may represent an assembly of protein, lipid, and pigment comprising all the elements of the photosystem II reaction. A scheme is presented which illustrates one possibility for the light reaction across the photosynthetic membrane.     

Single amino acid substitutions in the B870 alpha and beta light-harvesting polypeptides of Rhodobacter capsulatus. Structural and spectral effects.

To obtain information on the structural and functional role of highly conserved amino acid residues in the B870 alpha and beta light-harvesting polypeptides of Rhodobacter capsulatus, site-directed mutagenesis was performed. 18 mutants with single amino acid substitutions at nine different positions in the B870 antenna polypeptides were prepared in a B800-850-lacking strain. The characterization of the resulting phenotypes was based on a quantification of the core-complex elements (reaction center, light-harvesting polypeptides, bacteriochlorophyll a and carotenoid) and the core-complex spectral characteristics (absorption maximum, absorption coefficient and fluorescence intensity). These data generally showed that strong structural effects were caused by the amino acid substitutions. Thus, the three tryptophan exchanges at the position alpha 8 resulted in either the absence of a core complex (alpha Trp8----Leu), the absence of the core antenna (alpha Trp8----Ala) or a reduction in the carotenoid content (alpha Trp8----Tyr). Likewise, the mutants alpha Pro13Gly (i.e. alpha Pro13----Gly), beta Gly10Val and alpha Phe23Ala demonstrated an abnormal protein/pigment ratio in the core antenna, while a drastically reduced antenna size resulted from the amino acid exchange beta Arg45Asp. In contrast to the structural effects, the absorption maxima and the fluorescence intensities of the mutant antennae differed only slightly from the wild type. The strongest blue shift of the bacteriochlorophyll a (8-11 nm) was induced by substitutions of the Trp at position alpha 43 (alpha Trp43----Ala, Leu or Tyr). Contrary to the other spectral effects, the absorption coefficient of bacteriochlorophyll a was strongly influenced by the amino acid substitutions and varied by 1.6-times less (beta Arg45Asp) and 1.3-times greater (alpha Phe25Ala) than normal. The antenna-free mutant, alpha Trp8Ala, yielded a high rate of B800-850 revertants during phototrophic growth, indicating a direct energy transfer from the B800-850 antenna to the reaction center in these strains. Although conditions for growth were generally observed to influence phenotypic expression, the structural as well as spectral effects were demonstrated to differ to the greatest extent between chemotrophically grown and phototrophically grown cells.     

N-terminal sequences of subunits L and M of the photosynthetic reaction centre from Rhodospirillum rubrum G-9+. Separation of the subunits by gel filtration on hydroxypropylated Sephadex G 100 in organic solvents

A new method has been developed by which subunits L and M of the photosynthetic reaction centre from Rhodospirillum rubrum G-9+ can be obtained in pure form, starting form freeze-dried chromatophore membranes. The method employs extraction into a mixture of chloroform/methanol and gel permeation chromatography on a column of hydroxypropylated Sephadex G 100. Cross-contamination of the purified subunits was less than 5% (mol/mol), as estimated by manual Edman degradation. Automated Edman degradation has been carried out with both subunits in a liquid-phase sequencer. 36 amino-acid residues of subunit L and 50 residues of subunit M could be unequivocally identified. In both cases, the sequence analyses came to a premature end as the signal sudden by dropped to the level of the accidental fluctuations of the phenylthiohydantoin-derivatives background. This effect is explained by the unusual susceptibility to peptide bond cleavage of certain threonine residues which probably underwent N leads to O acyl shift during the cleavage reactions. The N-terminal sequences have been compared to those of subunits L and M of the photosynthetic reactions centre from Rhodopseudomonas sphaeroides R-26 (sutton, M.R., Rosen, D., Feher, G. & Steiner, L.A. (1982) Biochemistry 21, 3842-3849). The homology among subunits L is close to 90% and thus markedly higher than that among subunits M (32%). This finding indicates a pre-eminent role of subunit L in the primary events of photosynthetic energy conversion.     

Nucleotide and deduced polypeptide sequences of the photosynthetic reaction-center, B870 antenna, and flanking polypeptides from R. capsulata

The complete nucleotide sequence (8867 bp) and the deduced polypeptide sequence are given for 11 proteins from the photosynthetic gene cluster of R. capsulata (46 kb), including the photosynthetic reaction-center L, M, and H subunits and the B870 alpha and B870 beta polypeptides (light-harvesting I). These polypeptides bind bacteriochlorophyll, bacteriopheophytin, carotenoids, and quinones that are involved in the primary light reactions of photosynthesis. Hydropathy plots indicate that the L and M subunits are transmembrane proteins that may cross the membrane five times, while the H subunit has only one hydrophobic section near the amino terminus, which may be transmembrane. The L and M subunits are homologous over their entire length and have a high degree of homology with the QB protein from photosystem II of higher plants. An additional six genes were identified that may have some unknown role in bioenergetics since only mutations that affect the differentiation of the photosynthetic apparatus are known to map to this gene cluster.     

The membrane location of the B890-complex from Rhodospirillum rubrum and the effect of carotenoid on the conformation of its two apoproteins exposed at the cytoplasmic surface

The membrane location of the two B890-apoproteins from Rhodospirillum rubrum has been investigated by comparing the effects of mild proteolysis with proteinase K, and immunoprecipitation, with antibodies prepared against the B890-complex and its individual apoproteins, upon membrane vesicles. By using chromatophores (inside-out vesicles) and spheroplasts (right-side vesicles) both membrane surfaces have been interrogated. The N-terminal regions of both aproproteins are located at the cytoplasmic membrane surface, while the C-termini are most probably located at the periplasmic surface. The conformation of the N-terminal regions of the apoproteins is changed by the presence of carotenoid. In the carotenoid-containing strain S1, for example, the α-apoprotein is insensitive to digestion by proteinase K, while in the absence of the carotenoid in the mutant G9⁺ six amino acids are removed from the N-terminus of the α-aproprotein by proteinase K treatment.     

Photoreaction center of photosynthetic bacteria. 2. Size and quaternary structure of the photoreaction centers from Rhodospirillum rubrum strain G9 and from Rhodopseudomonas sphaeroides strain 2.4.1.

The photoreaction center from Rhodospirillum rubrum strain G9 binds about 6 times as much sodium dodecyl sulfate as certain proteins commonly used as molecular weight markers for sodium dodecyl sulfate--polyacrylamide gel electrophoresis. This presumably explains the apparent discrepancy between the molecular weight of the photoreaction center determined by electrophoresis (76 000) and its minimal molecular weight (87 000). The molecular weight of the photoreaction center solubilized with Triton X-100 was determined by three different methods: conventional sedimentation equilibrium, a combination of sedimentation velocity and gel filtration measurements, and sedimentation equilibrium in H2O and in D2O. Each technique required a determination of the amount of bound detergent. All three methods gave molecular weight values close to 60 000. A similar molecular weight was found for the photoactive beta gamma dimer obtained from the photoreaction center of Rhodopseudomonas sphaeroides strain 2.4.1 which, as a whole, had a molecular weight of 87 000. These results indicate that the photoreaction center from Rp. sphaeroides is an oligomer of the type alpha 1 beta 1 gamma 1. In contrast, the photoreaction center from Rs. rubrum appears to be dissociated, in solution, into a photoactive beta gamma dimer and a free polypeptide alpha.     

X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. I. Diffraction pattern of the photoreaction unit isolated from Rhodospirillum rubrum chromatophore and some characteristics of the structure

The X-ray diffraction pattern from chromatophore membranes of a photosynthetic bacterium, Rhodospirillum rubrum, indicates that a highly organized protein assembly exists in the membrane. The X-ray scatterer was solubilized from chromatophores by a mixture of cholate and deoxycholate. The basic component was identified as the photoreaction unit, which consists of light-harvesting bacteriochlorophyll proteins and a reaction center. The radial autocorrelation function, calculated directly from the X-ray intensity dats, made it possible to deduce certain structural features of the X-ray scatterer. 1. The maximum dimension of the X-ray scatterer is estimated to be 110-130 A. 2. The arrangement of the units in the chromatophore membrane is random. 3. Protein molecules in the unit form a rigid structure, being arranged mutually in fixed positions to give a distinct X-ray diffraction pattern. 4. The most probable structure is one which has rotational symmetry.     

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.     

Function of a PscD subunit in a homodimeric reaction center complex of the photosynthetic green sulfur bacterium Chlorobium tepidum studied by insertional gene inactivation. Regulation of energy transfer and ferredoxin-mediated NADP+ reduction on the cytoplasmic side

The PscD subunit in the homodimeric "type I" photosynthetic reaction center (RC) complex of the green sulfur bacterium Chlorobium tepidum was disrupted by insertional mutagenesis of its relevant pscD gene. This is the first report on the use of the direct mutagenic approach into the RC-related genes in green sulfur bacteria. The RC complex of C. tepidum is supposed to form a homodimer of two identical PscA subunits together with three other subunits: PscB (FA/FB-containing protein), PscC (cytochrome cz), and PscD. PscD shows a relatively low but significant similarity in its amino acid sequence to PsaD in the photosystem I of plants and cyanobacteria. We studied the biochemical and spectroscopic properties of a mutant lacking PscD in order to elucidate its unknown function. 1) The RC complex isolated from the mutant cells showed no band corresponding to PscD on SDS-PAGE analysis. 2) The growth rate of the PscD-less mutant was slower than that of the wild-type cells at low light intensities. 3) Time-resolved fluorescence spectra at 77 K revealed prolonged decay times of the fluorescence from bacteriochlorophyll c on the antenna chlorosome and from bacteriochlorophyll a on the Fenna-Matthews-Olson antenna protein in the mutant cells. The loss of PscD led to a much slower energy transfer from the antenna pigments to the special pair bacteriochlorophyll a (P840). 4) The mutant strain exhibited slightly less activity of ferredoxin-mediated NADP+ photoreduction compared with that in the wild-type strain. The extent of suppression, however, was less significant than that reported in the PsaD-less mutants of cyanobacterial photosystem I. The evolutionary relationship between PscD and PsaD was also discussed based on a structural homology modeling of the former.