The light-harvesting polypeptides of Rhodospirillum rubrum. II. Localisation of the amino-terminal regions of the light-harvesting polypeptides B 870-alpha and B 870-beta and the reaction-centre subunit L at the cytoplasmic side of the photosynthetic membrane of Rhodospirillum rubrum G-9+

The unspecific proteinase K and the specific proteases alpha-chymotrypsin, trypsin and S. aureus V 8 protease were used in order to determine the orientation of the polypeptides B 870-alpha and B 870-beta from the major antenna complex B 870 of Rs. rubrum G-9+ within the chromatophore membrane (inside-out vesicle). Although B 870-alpha exhibits cleavable peptide bonds, treatment with specific proteases yielded splitting only in B 870-beta within the N-terminal region. In the case of proteinase K, which was most effective, mainly 6 (B 870-alpha) and 16 (B 870-beta) amino acid residues were removed from their N-terminal parts as proved by means of Edman degradation of cleavage products. The major peptide bonds cleaved were identified as Gln6-Leu7 in B 870-alpha and as Lys16-Glu17 in B 870-beta. The central hydrophobic stretch regions and the relatively hydrophilic C-terminal parts of both light-harvesting polypeptides were not affected by proteinase K. On the basis of these degradation experiments a transmembrane orientation of B 870-alpha and B 870-beta is postulated, with their N-terminal towards the cytoplasm and their C-termini towards periplasm with regard to the photosynthetic membrane. This hypothesis is supported by the transmembrane model proposed by Brunisholz et al. (Hoppe-Seyler's Z., Physiol. Chem., (1984) 365, 675-688) in which the hydrophobic stretch of B 870-alpha and of B 870-beta forming an alpha-helix would span the membrane once. Organic solvent extraction of chromatophores treated with proteinase K yielded a fairly pure polypeptide fragment with an apparent molecular mass of 14000 Da. Its N-terminal amino-acid sequence is identical with the sequence within the N-terminal region of the reaction centre subunit L of Rs. rubrum G-9+. Thus it is most likely that as in the case of B 870-beta, proteinase K removed 16 amino acid residues from the N-terminal part of subunit L. This subunit therefore also seems to be exposed at the surface of the cytoplasmic side of the chromatophore membrane.     

Localization of reaction center and B800-850 antenna pigment proteins in membranes of Rhodobacter sphaeroides.

The localization of the N- and C-terminal regions of pigment-binding polypeptides of the bacterial photosynthetic apparatus of Rhodobacter sphaeroides was investigated by proteinase K treatment of chromatophore and spheroplast-derived vesicles and amino acid sequence determination. Under conditions of proteinase K treatment of chromatophores, which left the in vivo absorption spectrum and the membrane intact, 15 and 46 amino acyl residues from the N-terminal regions of the L and M subunits, respectively, of the reaction center polypeptides were removed. The N termini are therefore exposed on the cytoplasmic surface of the membrane. The C-terminal domain of the light-harvesting B800-850 alpha and B870 alpha polypeptides was found to be exposed on the periplasmic surface of the membrane. A total of 9 and 13 amino acyl residues were cleaved from the B800-850 alpha and B870 alpha polypeptides, respectively, when spheroplasts were treated with proteinase K. The N-terminal regions of the alpha polypeptides were not digested in either membrane preparation and were apparently protected from proteolytic attack. Seven N-terminal amino acyl residues of the B800-850 beta polypeptide were removed after the digestion of chromatophores. C-terminal residues were not removed after the digestion of chromatophores or spheroplasts. The C termini seem to be protected from protease attack by interaction with the membrane. Therefore, the N-terminal regions of the beta polypeptides are exposed on the cytoplasmic membrane surface. The C termini of the beta polypeptides are believed to point to the periplasmic space.     

Amino-terminal sequences of the L, M, and H subunits of reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26

We have determined the sequence of the 25-28 amino-terminal residues of the three subunits, L, M, and H, of the membrane-bound reaction center protein of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26. The sequences are as follows: L, H2N-Ala-Leu-Leu-Ser-Phe-Glu-Arg-Lys-Tyr-Arg- Val-Pro-Gly-Gly-Thr-Leu-Val-Gly-Gly-Asn-Leu-Phe-Asp-Phe-(His)-Val-; M, H2N-Ala-Glu-Tyr-Gln-Asn-Ile-Phe-Ser-Gln-Val-Gln-Val-Arg-Gly-Pro-Ala-Asp-Leu-Gly-Met-Thr-Glu-Asp-Val-Asn-Leu-Ala-Asn-; H, H2N-Met-Val-Gly-Val-Thr-Ala-Phe-Gly-Asn-Phe-Asp-Leu-Ala-Ser-Leu-Ala-Ile-Tyr-Ser-Phe-Trp-Ile-Phe-Leu-Ala-X-Leu-Ile-. The H sequence, especially after the aspartyl residue at position 11, is rich in hydrophobic residues, consistent with the possibility that this section of the polypeptide chain is located within the membrane. The L sequence is hydrophilic near the amino terminus and then becomes moderately hydrophobic. The M sequence is of average polarity.     

Analysis of genomic diversity among photosynthetic stem-nodulating rhizobial strains from northeast Argentina

The genomic diversity among photosynthetic rhizobia from northeast Argentina was assessed. Forty six isolates obtained from naturally occurring stem and root nodules of Aeschynomene rudis plants were analyzed by three molecular typing methods with different levels of taxonomic resolution: repetitive sequence-based PCR (rep-PCR) genomic fingerprinting with BOX and REP primers, amplified 16S rDNA restriction analysis (ARDRA), and 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (IGS-RFLP) analysis. The in vivo absorption spectra of membranes of strains were similar in the near infrared region with peaks at 870 and 800 nm revealing the presence of light harvesting complex I, bacteriochlorophyll-binding polypeptides (LHI-Bchl complex). After extraction with acetone-methanol the spectra differed in the visible part displaying peaks belonging to canthaxanthin or spirilloxanthin as the main carotenoid complement. The genotypic characterization by rep-PCR revealed a high level of genomic diversity among the isolates and almost all the photosynthetic ones have identical ARDRA patterns and fell into one cluster different from Bradyrhizobium japonicum and Bradyrhizobium elkanii. In the combined analysis of ARDRA and rep-PCR fingerprints, 7 clusters were found including most of the isolates. Five of those contained only photosynthetic isolates; all canthaxanthin-containing strains grouped in one cluster, most of the other photosynthetic isolates were grouped in a second large cluster, while the remaining three clusters contained a few strains. The other two clusters comprising reference strains of B. japonicum and B. elkanii, respectively. The IGS-RFLP analysis produced similar clustering for almost all the strains. The 16S rRNA gene sequence of one representative isolate was determined and the DNA sequence analysis confirmed the position of photosynthetic rhizobia in a distinct phylogenetic group within the Bradyrhizobium rDNA cluster.     

Structural aspects of proton-pumping ATPases

ATP synthase is found in bacteria, chloroplasts and mitochondria. The simplest known example of such an enzyme is that in the eubacterium Escherichia coli; it is a membrane-bound assembly of eight different polypeptides assembled with a stoichiometry of alpha 3 beta 3 gamma 1 delta 1 epsilon 1 a1b2c10-12. The first five of these constitute a globular structure, F1-ATPase, which is bound to an intrinsic membrane domain, F0, an assembly of the three remaining subunits. ATP synthases driven by photosynthesis are slightly more complex. In chloroplasts, and probably in photosynthetic bacteria, they have nine subunits, all homologues of the components of the E. coli enzyme; the additional subunit is a duplicated and diverged relation of subunit b. The mammalian mitochondrial enzyme is more complex. It contains 14 different polypeptides, of which 13 have been characterized. Two membrane components, a (or ATPase-6) and A6L, are encoded in the mitochondrial genome in overlapping genes and the remaining subunits are nuclear gene products that are translated on cytoplasmic ribosomes and then imported into the organelle. The sequence of the proteins of ATP-synthase have provided information about amino acids that are important for its function. For example, amino acids contributing to nucleotide binding sites have been identified. Also, they provide the basis of models of secondary structure of membrane components that constitute the transmembrane proton channel. An understanding of the coupling of the transmembrane potential gradient for protons, delta mu H+, to ATP synthesis will probably require the determination of the structure of the entire membrane bound complex. Crystals have been obtained of the globular domain, F1-ATPase. They diffract to a resolution of 3-4 A and data collection is in progress. As a preliminary step towards crystallization of the entire complex, we have purified it from bovine mitochondria and reconstituted it into phospholipid vesicles.     

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.     

The photosynthesis gene cluster of Rhodobacter sphaeroides

The photosynthetic bacteria are at the forefront of the study of many aspects of photosynthesis, including photopigment biosynthesis, photosynthetic-membrane assembly, light-harvesting, and reaction center photochemistry. The facultative growth of some photosynthetic bacteria, their simple photosystems, and their ease of genetic manipulation have all contributed to advances in these areas. Amongst these bacteria, the purple non-sulfur bacterium Rhodobacter sphaeroides has emerged as, arguably, the leading contender for a model system in which to integrate the studies of all the different aspects of the assembly and function of the photosynthetic apparatus. Many of the genes encoding photosynthesis-related proteins are known to be clustered within a small region of the genome in this organism. As a further aid to studying the assembly and function of the photosystem of Rb. sphaeroides, the DNA sequence for a genomic segment containing this photosynthesis gene cluster (PGC) has been assembled from previous EMBL submissions and formerly unpublished data. The Rb. sphaeroides PGC is 40.7 kb in length and consists of 38 open reading frames encoding the reaction center H, L and M subunits, the and polypeptides of the light-harvesting I (B875) complex, and the enzymes of bacteriochlorophyll and carotenoid biosynthesis. PGCs are a feature of gene organization in several photosynthetic bacteria, and the similarities between the clusters of Rb. sphaeroides and Rb. capsulatus have been apparent for some time. Here we present the first comprehensive analysis of the PGC of Rb. sphaeroides, as well as a comparison with that of Rb. capsulatus.     

Topography of reaction center subunits in the membrane of the photosynthetic bacterium, rhodopseudomonas sphaeroides

The localization of the reaction center polypeptides (L, M, and H) in the membranes of both the wild-type, strain 2.4.1, and the carotenoidless mutant, R-26, of Rhodopseudomonas sphaeroides was determined by using affinity-purified antibodies specific for these proteins. Binding of the antibodies to reaction center subunits in spheroplasts was visualized in the electron microscope by immunoferritin labeling. The H and M subunits were labeled at both the cytoplasmic and the periplasmic surfaces of the membrane, whereas the L subunit was labeled only at the periplasmic surface of the membrane. Thus, the reaction center is asymmetrically oriented in the membrane with at least two subunits (H and M) spanning the membrane.     

A three-dimensional model of the Photosystem II reaction centre of Pisum sativum

A three-dimensional model of the core proteins D1 and D2, including the cofactors, that form the Photosystem II reaction centre of pea (Pisum sativum), has been generated. This model was built with a rule-based computer modelling system using the information from the crystal structures of the photosynthetic reaction centres of Rhodopseudomonas viridis and Rhodobacter sphaeroides. An alignment of the primary sequences of twenty three D1, nine D2, eight bacterial L and eight bacterial M subunits predicts strong similarity between bacterial and higher plant reaction centres, especially in the transmembrane region where the cofactors responsible for electron transport are located. The sequence to be modelled was aligned to the bacterial structures using environment-dependent substitution tables to construct matrices, improving the alignment procedure. The ancestral relationship between the bacteria and higher plant sequences allowed both the L and M subunits to be used as structural templates as they were equally related to the higher plant polypeptides. The regions with the highest predicted structural homology were used as a framework for the construction of the structurally conserved regions. The structurally conserved region of the model shows strong similarity to the bacterial reaction centre in the transmembrane helices. The stromal and lumenal loops show greater sequence variation and are therefore predicted to be the structurally variable regions in the model. The key sidechain assignments and residues that may interact with cofactors are discussed.Abbreviations D Tyr161 in the D2 polypeptide - PS II Photosystem II - Q_A primary plastoquinone acceptor of Photosystem II - Q_B secondary plastoquinone acceptor of Photosystem II - Z Tyr161 in the D1 polypeptide     

Primary structure of the reaction center from Rhodopseudomonas sphaeroides

The reaction center is a pigment-protein complex that mediates the initial photochemical steps of photosynthesis. The amino-terminal sequences of the L, M, and H subunits and the nucleotide and derived amino acid sequences of the L and M structural genes from Rhodopseudomonas sphaeroides have previously been determined. We report here the sequence of the H subunit, completing the primary structure determination of the reaction center from R. sphaeroides. The nucleotide sequence of the gene encoding the H subunit was determined by the dideoxy method after subcloning fragments into single-stranded M13 phage vectors. This information was used to derive the amino acid sequence of the corresponding polypeptide. The termini of the primary structure of the H subunit were established by means of the amino and carboxy terminal sequences of the polypeptide. The data showed that the H subunit is composed of 260 residues, corresponding to a molecular weight of 28,003. A molecular weight of 100,858 for the reaction center was calculated from the primary structures of the subunits and the cofactors. Examination of the genes encoding the reaction center shows that the codon usage is strongly biased towards codons ending in G and C. Hydropathy analysis of the H subunit sequence reveals one stretch of hydrophobic residues near the amino terminus; the L and M subunits contain five such stretches. From a comparison of the sequences of homologous proteins found in bacterial reaction centers and photosystem II of plants, an evolutionary tree was constructed. The analysis of evolutionary relationships showed that the L and M subunits of reaction centers and the D1 and D2 proteins of photosystem II are descended from a common ancestor, and that the rate of change in these proteins was much higher in the first billion years after the divergence of the reaction center and photosystem II than in the subsequent billion years represented by the divergence of the species containing these proteins.     

Biochemical and immunological studies of bovine and porcine neurofilament triplet proteins by peptide mapping after cyanogen bromide cleavage

Peptide mapping of the three bovine and porcine neurofilament protein subunits ("L", "M" and "H") with apparent mol. wts of 70, 160 and 210 kDa were performed with CNBr, leading to the cleavage of methionyl bonds. We have obtained two characteristic large fragments with molecular weights of 85 kDa for the "M" bovine subunit and 135 kDa for the "H" subunit of bovine neurofilament. A comparison of the electrophoretic patterns of CNBr generated polypeptides of "L" subunit from beef and pig showed that they are highly related structures. The peptide mappings of CNBr peptides of "M" and "H" subunits from beef and pig were significantly different. Antibodies were raised against the 85 kDa and 135 kDa CNBr fragments. Immunoblotting results with anti-85 kDa and anti-135 kDa of beef are in favour of large differences of structure between the "M" subunits from pig and beef. The "H" proteins were very similar and they also showed that the C-terminal part of bovine "H" and "M" proteins share common antigenic determinants.     

The amino-acid sequence of the 2S sulphur-rich proteins from seeds of Brazil nut (Bertholletia excelsa H.B.K.)

Storage proteins of the albumin solubility fraction from seeds of Bertholletia excelsa H.B.K. were separated by reversed-phase high-performance liquid chromatography and their primary structures were determined by gas-phase sequencing on intact polypeptides and on the overlapping tryptic and thermolysin peptides. The 2S storage proteins consist of two subunits linked by disulphide bridges. The large subunit (8.5 kDa) is expressed in at least six different isoforms while the small subunit (3.6 kDa) consists of only one form. These proteins are extremely rich in glutamine, glutamic acid, arginine and the sulphur-containing amino acids cysteine and methionine. One of the variants even contains a sequence of six methionine residues in a row. Comparison with known sequences of 2S proteins of other dicotyledonous plants shows limited but distinct sequence homology. In particular, the positions of the cysteine residues relative to each other appear to be completely conserved, suggesting that tertiary structure constraints imposed by disulphide bridges dominate sequence conservation. It has been proposed that the two subunits of a related protein (the Brassica napus storage protein) is cleaved from a precursor polypeptide [Crouch, M. L., Tenbarge, K. M., Simon, A. E. & Ferl, R. (1983) J. Mol. Appl. Genet. 2,273-283]. The amino acid sequence homology of the Brazil nut protein with the former suggests that a similar protein processing event could occur.     

Isolation of a gene that encodes a new retinal protein, archaerhodopsin, from Halobacterium sp. aus-1

We have cloned and sequenced the gene that encodes archaerhodopsin, a light-driven H+ pump in Halobacterium sp. aus-1 (Mukohata, Y., Sugiyama, Y., Ihara, K., and Yoshida, M. (1988) Biochem. Biophys. Res. Commun. 151, 1339-1345). The nucleotide sequence of this gene contained an open reading frame which corresponded to a protein of 260 amino acids with a molecular mass of 27,851 daltons, including a precursor sequence of 6 amino acids at the amino terminus and 2 amino acids at the carboxyl terminus. The deduced amino acid sequence of archaerhodopsin exhibited 59 and 32% homology to the sequences of bacteriorhodopsin and halorhodopsin, respectively, from Halobacterium halobium. Three charged residues (Asp-121, Asp-218, and Lys-222) are conserved in the transmembrane segments among the three retinal proteins. Residues Asp-91 and Asp-102 which, it has been suggested, may be essential for the pumping of protons (Mogi, T., Stern, L. J., Marti, T., Chao, B. H., and Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,4148-4152) are conserved between archaerhodopsin and bacteriorhodopsin.     

Characterization of the formyltransferase from Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 possesses a formaldehyde-oxidation pathway that involves enzymes with high sequence identity with enzymes from methanogenic and sulfate-reducing archaea. Here we describe the purification and characterization of formylmethanofuran-tetrahydromethanopterin formyltransferase (Ftr), which catalyzes the reversible formation of formylmethanofuran (formylMFR) and tetrahydromethanopterin (H4MPT) from N5-formylH4MPT and methanofuran (MFR). Formyltransferase from M. extorquens AM1 showed activity with MFR and H4MPT isolated from the methanogenic archaeon Methanothermobacter marburgensis (apparent Km for formylMFR = 50 microM; apparent Km for H4MPT = 30 microM). The enzyme is encoded by the ffsA gene and exhibits a sequence identity of approximately 40% with Ftr from methanogenic and sulfate-reducing archaea. The 32-kDa Ftr protein from M. extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa. Interestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively. The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M. extorquens AM1 indicates that, in the bacterium, the respective polypeptides form a functional unit. Expression studies in Escherichia coli indicate that Ftr requires the other subunits of the complex for stability. Despite the fact that three of the polypeptides of the complex showed sequence similarity to subunits of Fmd from methanogens, the complex was not found to catalyze the oxidation of formylMFR. Detailed comparison of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the active-site cofactors molybdenum, molybdopterin and a [4Fe-4S] cluster. Cytochrome c was found to be spontaneously reduced by H4MPT. On the basis of this property, a novel assay for Ftr activity and MFR is described.