Soluble cytochromes and a photoactive yellow protein isolated from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salexigens.

Three soluble cytochromes were found in two strains of the halophilic non-sulfur purple bacterium Rhodospirillum salexigens. These are cytochromes C2, C and c-551. Cytochrome C2 was recognized by the presence of positive charge at the site of electron transfer (measured by laser flash photolysis), although the protein has an overall negative charge (pI = 4.7). Cytochrome C2 has a high redox potential (300 mV) and is monomeric (13 kDa). Cytochrome c was recognized from its characteristic absorption spectrum. It has a redox potential of 95 mV, an isoelectric point of 4.3, and is isolated as a dimer (33 kDa) of identical subunits (14 kDa), a property which is typical of this family of proteins. R. salexigens cytochrome c-551 has an absorption spectrum similar to the low redox potential Rb. sphaeroides cytochrome c-551.5. It also has a low redox potential (-170 mV), is very acidic (pI = 4.5), and is monomeric (9 kDa), apparently containing 1 heme per protein. The existence of abundant membrane-bound cytochromes c-558 and c-551 which are approximately half reduced by ascorbate and completely reduced by dithionite suggests the presence of a tetraheme reaction center cytochrome in R. salexigens, although reaction centers purified in a previous study (Wacker et al., Biochim. Biophys. Acta (1988) 933, 299-305) did not contain a cytochrome. The most interesting observation is that R. salexigens contains a photoactive yellow protein (PYP), previously observed only in the extremely halophilic purple sulfur bacterium Ectothiorhodospira halophila. The R. salexigens PYP appears to be slightly larger than that of Ec. halophila (16 kDa vs. 14 kDa). Otherwise, these two yellow proteins have similar absorption spectra, chromatographic properties and kinetics of photobleaching and recovery.

Structure of ferricytochrome c' from Rhodospirillum molischianum at 1.67 A resolution.

The structure of ferricytochrome c' from Rhodospirillum molischianum has been crystallographically refined to 1.67 A resolution using a combination of reciprocal space and restrained least-squares refinement methods. The final crystallographic R-factor for 30,533 reflections measured with I greater than sigma (I) between infinity and 1.67 A is 0.188. The final model incorporates 1944 unique protein atoms (of a total of 1972) together with 194 bound solvent molecules. The structure has been analysed with respect to its detailed conformational properties, secondary structural features, temperature factor behavior, bound solvent sites, and heme geometry. The asymmetric unit of the cytochrome c' crystal contains a dimer composed of chemically identical 128-residue polypeptide chains. Although the refined structure shows the monomers to be very similar, examination of the differences that do occur allows an evaluation of how different lattice contacts affect protein conformation and solvent binding. In particular, comparison of solvent binding sites in the two subunits allows identification of a common set that are not altered by lattice interactions. The preservation of these solvent interactions in different lattice environments suggests that they play a structural role in protein stabilization in solution. The refined structure additionally reveals some new features that relate to the ligand binding properties and unusual mixed-spin state character of cytochrome c'. Finally, comparison of the heme binding geometry in cytochrome c' and other structurally unrelated c-type cytochromes shows that two alternative, but sterically favorable, conformational variants occur among the seven examples examined.

The amino acid sequence of a high-redox-potential ferredoxin from the purple phototrophic bacterium, Rhodospirillum tenue strain 2761.

The 61-residue amino acid sequence of Rhodospirillum tenue, strain 2761, high-redox-potential ferredoxin (HiPIP) is GTNAAMRKAFNYQDTAKNGKCSGCAQFVPGASPTAAGGCKVIPGDNEIAPGGYCDAFIVKK. It differs from that of R. tenue strain 3761 by 16 amino acid substitutions plus two single-residue deletions. This 26% sequence difference is similar to that observed among separate species of chromatiaceae such as Chromatium vinosum, C. gracile, and Thiocapsa roseopersicina, and is suprising because there are no distinguishing microbiological characteristics separating these two R. tenue strains. The most interesting amino acid substitution in R. tenue 2761 HiPIP is Gly for Asn 45 (C. vinosum numbering). Besides the four cysteines required to bind the four iron-four sulfur cluster, only Tyr 19, Asn 45, and Gly 75 are absolutely conserved in the nine previously determined HiPIP sequences. If HiPIP is used as a measure of divergence of species, then R. tenue and C. vinosum are the most distant purple bacteria examined. Quite the opposite conclusion follows based on the sequences of the cytochromes c'. It is suggested that this anomaly is more likely owing to a change in function for HiPIP with subsequently rapid evolutionary change than to a relatively recent transfer of the cytochrome c' gene between species.

Nature and location of amide-bound (R)-3-acyloxyacyl groups in lipid A of lipopolysaccharides from various gram-negative bacteria.

It has previously been demonstrated [Eur. J. Biochem. 124, 191-198 (1982) and 137, 15-22 (1983)] that the lipid A component of Salmonella and Proteus lipopolysaccharides contains amide-linked (R)-3-acyloxyacyl residues. In the present study lipid A of other gram-negative bacteria was analysed for the presence of amide-bound 3-acyloxyacyl residues. It was found that such residues are constituents of all lipid A tested (Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeruginosa, Xanthomonas sinensis, Bacteroides fragilis, Vibrio cholerae, Fusobacterium nucleatum, Rhodospirillum tenue, Acinetobacter calcoaceticus, and Escherichia coli). Amide-linked (R)-3-acyloxyacyl groups, therefore, represent common and ubiquitous structural elements of bacterial lipid A. The composition of 3-acyloxyacyl groups differed considerably among different bacteria. As amide-bound (R)-3-hydroxy fatty acids straight chain and isobranched acyl groups with 10-17 carbon atoms were identified. The most frequently encountered fatty acids, substituting the 3-hydroxyl group of 3-hydroxy fatty acids, were nonhydroxylated straight chain and isobranched acyl residues with 10-17 carbon atoms as well as (S)-2-hydroxy fatty acids with 12 carbon atoms. In some cases, using laser desorption mass spectrometry, the distribution of 3-acyloxyacyl residues over the two available glucosamine amino groups of the lipid A backbone was investigated.

Protein on the cell surface of the moderately halophilic phototrophic bacterium Rhodospirillum salexigens.

A cell surface protein (Mr 68,000) of the moderately but obligately halophilic phototrophic bacterium Rhodospirillum salexigens was identified by two independent methods: first, by labeling the cell surface with radioactive iodine and lactoperoxidase, and second, by washing cells in 30% sucrose to remove proteins attached to the cell surface by ionic bonds. The identified protein very likely represents the outermost layer of the cell envelope of R. salexigens as observed by electron microscopy. The protein was isolated. Its isoelectric point was determined to be 4.4; the excess of acidic over basic amino acids was found to be 18.3 mol%; and its average hydrophobicity was 2.26 kJ per residue.

Oxido-reduction of B800-850 and B880 holochromes isolated from three species of photosynthetic bacteria as studied by electron-paramagnetic resonance and optical spectroscopy.

Certain redox properties of bacteriochlorophyll alpha were used to probe the structure of several light-harvesting pigment-protein complexes or holochromes. To attribute redox properties unequivocally to a given holochrome, we worked with purified holochromes. We developed purification procedures for the B880 holochromes from Rhodospirillum rubrum, Rhodopseudomonas sphaeroides and Ectothiorhodospira sp. and for the B800-850 holochromes from the latter two species. In all these holochromes, bacteriochlorophyll alpha could be oxidized by ferricyanide as witnessed by the bleaching of their near-infrared absorption bands. However, only in B880 holochromes was this oxidation reversible. Another important difference between the B800-850 and the B880 holochromes is that oxidation of the latter gives rise to a g = 2.0025 electron paramagnetic resonance (EPR) signal with linewidth varying, according to species, from 0.37 mT to 0.48 mT. Both the reversible EPR signal and absorption changes titrate with a midpoint redox potential (pH 8.0) of approximately 570 mV. Linewidth narrowing can be interpreted by delocalization of the free electron spin over approximately 12 bacteriochlorophyll molecules. While the B880 holochromes from the three species considered had indistinguishable redox properties, the B800-850 holochromes differed from one another by their circular dichroic spectra and by the relative ease of oxidation of their 800-nm and 850-nm bands. This indicates that, contrary to the B880 holochromes, the B800-850 holochromes may not form a homogeneous class.

Polar lipids in phototrophic bacteria of the Rhodospirillaceae and Chromatiaceae families.

The polar lipids of photosynthetic purple bacteria of the genera Chromatium, Thiocapsa, Thiocystis, Ectothiorhodospira, Rhodopseudomonas, Rhodospirillum, and Rhodomicrobium were analyzed. Characteristic compositions of the polar lipids were found for most of the Rhodospirillaceae and Chromatiaceae species. Phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin were the major phospholipids in most species. Phosphatidylcholine was present as a major component in all species of the genus Ectothiorhodospira, but was not detected in the remaining Chromatiaceae. It was also present in most of the Rhodospirillaceae species. No glycolipids were found in any of the Ectothiorhodospira species. In the Rhodospirillaceae, the glycolipids mono- and digalactosyl diglycerides were generally absent. Sulfoquinovosyl diglyceride was present in significant amounts in at least three species of the Rhodospirillaceae and may have been present in most of them, but only in traces. All of the Chromatiaceae species contained several glycolipids, one of which was similar to monogalactosyl diglyceride. Ornithine lipids were found in large amounts in most Rhodospirillaceae, but were absent in Ectothiorhodospira and in the other Chromatiaceae. The species examined could be divided into three groups on the basis of their lipid composition: (i) the genus Ectothiorhodospira; (ii) the remaining Chromatiaceae; and (iii) the Rhodospirillaceae. The data presented are compared with those available in the literature, and differences from other phototrophic organisms are discussed.

Structure of the heptose region of lipopolysaccharies from Rhodospirillum tenue.

There is a common structure (core region) in the lipopolysaccharides of Rhodospirillum tenue. It is composed of a branched trisaccharide of L-glycero-D-mannoheptose (and of 2-keto-3-deoxyoctonate), as revealed by methylation analyses of degraded polysaccharides of four different R. tenue strains. The structure is similar or might even be identical to the inner core of enterobacterial O antigens. In addition, each of the four R. tenue lipopolysaccharides contains a strain-specific region that consists of heptose(s) (L-glycero-D-mannoheptose or D-glycero-D-mannoheptose or both) or hexoses. There is a partial substitution of the core region and the strain-specific region by phosphorus, showing microheterogeneity.

Primary structure of a high potential, four-iron-sulfur ferredoxin from the photosynthetic bacterium Rhodospirillum tenue.

The amino acid sequence of a high oxidation-reduction potential iron-sulfur protein (HiPIP) isolated from the purple photosynthetic bacterium Rhodospirillum tenue has been determined. This is the smallest of the HiPIP's, containing 63 residues, with only 3 residues apparently conserved in addition to the 4 cluster-binding cysteines. A minimum of four internal genetic gaps is postulated to align tenue high potential iron-sulfur protein with the previously known proteins. It is the most divergent of its class, with an average of only 25% identically matching residues in common with any of the other species.