GlbN (cyanoglobin) is a peripheral membrane protein that is restricted to certain Nostoc spp.

The glbN gene of Nostoc commune UTEX 584 is juxtaposed to nifU and nifH, and it encodes a 12-kDa monomeric hemoglobin that binds oxygen with high affinity. In N. commune UTEX 584, maximum accumulation of GlbN occurred in both the heterocysts and vegetative cells of nitrogen-fixing cultures when the rate of oxygen evolution was repressed to less than 25 micromol of O2 mg of chlorophyll a(-1) h(-1). Accumulation of GlbN coincided with maximum synthesis of NifH and ferredoxin NADP+ oxidoreductase (PetH or FNR). A total of 41 strains of cyanobacteria, including 40 nitrogen fixers and representing 16 genera within all five sections of the cyanobacteria were screened for the presence of glbN or GlbN. glbN was present in five Nostoc strains in a single copy. Genomic DNAs from 11 other Nostoc and Anabaena strains, including Anabaena sp. strain PCC 7120, provided no hybridization signals with a glbN probe. A constitutively expressed, 18-kDa protein which cross-reacted strongly with GlbN antibodies was detected in four Anabaena and Nostoc strains and in Trichodesmium thiebautii. The nifU-nifH intergenic region of Nostoc sp. strain MUN 8820 was sequenced (1,229 bp) and was approximately 95% identical to the equivalent region in N. commune UTEX 584. Each strand of the DNA from the nifU-nifH intergenic regions of both strains has the potential to fold into secondary structures in which more than 50% of the bases are internally paired. Mobility shift assays confirmed that NtcA (BifA) bound a site in the nifU-glbN intergenic region of N. commune UTEX 584 approximately 100 bases upstream from the translation initiation site of glbN. This site showed extensive sequence similarity with the promoter region of glnA from Synechococcus sp. strain PCC 7942. In vivo, GlbN had a specific and prominent subcellular location around the periphery of the cytosolic face of the cell membrane, and the protein was found solely in the soluble fraction of cell extracts. Our hypothesis is that GlbN scavenges oxygen for and is a component of a membrane-associated microaerobically induced terminal cytochrome oxidase.     

Spectroscopical and functional characterization of the hemoglobin of Nostoc commune UTEX 584 (Cyanobacteria)

Structural analysis of a monomeric hemoglobin from the cyanobacterium Nostoc commune strain UTEX 584, cyanoglobin (Potts et al. (1992) Science 256, 1690–1692), is presented. Cyanoglobin binds molecular oxygen reversibly, with high oxygen affinity and non-cooperativity. There was no evidence for decreased stability of the pigment at 37°C. Cyanoglobin-specific antibodies showed no cross-reactivity with two reference hemoglobins, leghemoglobin a and sperm whale myoglobin. The absorption spectral properties of cyanoglobin differ significantly from those of the two reference hemoglobins. The spectrum of oxy-cyanoglobin most closely resembles that of an oxy-hemoglobin from the protozoan Tetrahymena pyriformis, a hemoprotein that shares substantial amino-acid sequence identity with cyanoglobin. Met-cyanoglobin possesses spectral characteristics at pH 7.0–9.0 that resemble those of the alkaline met-hemoglobin (a putative hemichrome) of another protozoan, Paramecium caudatum. The spin-state character of met-cyanoglobin is pH-dependent. Met-cyanoglobin does not coordinate the strong-field ligands, cyanide and azide, at pH 7.0. The capacity of cyanoglobin to coordinate cyanide increased with decreasing pH. Far-UV CD spectra of cyanoglobin are indicative of a protein with a significant amount of alpha-helical structure. Data from Soret-region CD spectra suggest that the orientations of the heme moieties in cyanoglobin and leghemoglobin a are similar to one another.     

Cyanobacterial RNA polymerase genes rpoC1 and rpoC2 correspond to rpoC of Escherichia coli.

The DNA-dependent RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of cyanobacteria contains a unique core component, gamma, which is absent from the RNA polymerases of other eubacteria (G. J. Schneider, N. E. Tumer, C. Richaud, G. Borbely, and R. Haselkorn, J. Biol. Chem. 262:14633-14639, 1987). We present the complete nucleotide sequence of rpoC1, the gene encoding the gamma subunit, from the heterocystous cyanobacterium Nostoc commune UTEX 584. The derived amino acid sequence of gamma (621 residues) corresponds with the amino-terminal portion of the beta' polypeptide of Escherichia coli RNA polymerase. A second gene in N. commune UTEX 584, rpoC2, encodes a protein which shows correspondence with the carboxy-terminal portion of the E. coli beta' subunit. The rpoBC1C2 genes of N. commune UTEX 584 are present in single copies and are arranged in the order rpoBC1C2, and the coding regions are separated by short AT-rich spacer regions which have the potential to form very stable secondary structures. Our data indicate the occurrence of divergent evolution of structure in the eubacterial DNA-dependent RNA polymerase.     

Cloning of nifHD from Nostoc commune UTEX 584 and of a flanking region homologous to part of the Azotobacter vinelandii nifU gene.

The heterocystous cyanobacterium Nostoc commune UTEX 584 contains two nifH-like sequences (nifH1 and nifH2) in addition to nifHD. A region of DNA 1 kilobase upstream from the 5' end of nifH showed considerable sequence similarity to part of the published nifU sequences of Azotobacter vinelandii and Klebsiella pneumoniae.     

Polysome turnover in immobilized cells of Nostoc commune (cyanobacteria) exposed to water stress.

Water stress induced changes in the polysome content of immobilized cells of the desiccation-tolerant cyanobacterium Nostoc commune UTEX 584. Cells maintained an intact protein synthesis complex during 2 h of drying at -99.5 MPa. Polysomes were not recovered from cells subjected to extended periods of desiccation.     

In vitro translation of mRNA from Nostoc commune (Cyanobacteria)

RNA pools were extracted from cells of Nostoc commune UTEX 584 in exponential growth (liquid cultures) and from cells which had been immobilized and dried rapidly at -99.5 MPa. Levels of incorporation of ³⁵S-methionine, five- to sixfold higher than the endogenous level, were obtained after in vitro translation of the RNA preparations in a heterologous S30 cell-free system purified from Escherichia coli Q13. The levels of incorporation, obtained with a homologous N. commune UTEX 584 S30 system, were much lower. The requirement for magnesium in the heterologous system was 15–21 mM, translation of N. commune UTEX 584 RNA was inhibited when the RNA concentration was greater than 0.3 mg ml^–1, and translation was stimulated significantly by the presence of ammonium chloride. Few qualitative differences were observed between the pattern of proteins (SDS-PAGE) obtained after translation of the RNA pools from cells in exponential growth, and from those cells subjected to immobilization and rapid drying. The data suggest that short-term desiccation of N. commune UTEX 584 does not have a marked selective effect on the composition of the mRNA pool. In contrast, preparations of RNA from field materials of Nostoc commune HUN (desiccated for 5 years) were unable to drive high rates of translation in any of the systems tested and optimized for use in this study.     

Protein synthesis and proteolysis in immobilized cells of the cyanobacterium Nostoc commune UTEX 584 exposed to matric water stress.

Cells of the cyanobacterium Nostoc commune UTEX 584 in exponential growth were subjected to acute water stress by immobilizing them on solid supports and drying them at a matric water potential (psi m) of -99.5 MPa. Cells which had been grown in the presence of Na235SO4 before immobilization and rapid drying continued to incorporate 35S into protein for 90 min. This incorporation was inhibited by chloramphenicol. No unique proteins appeared to be synthesized during this time. Upon further drying, the level of incorporation of 35S in protein began to decrease. In contrast, there was an apparent increase in the level of certain phycobiliprotein subunits in solubilized protein extracts of these cells. Extensive proteolysis was detected after prolonged desiccation (17 days) of the cells in the light, although they still remained intact. Phycobilisomes became dissociated in both light- and dark-stored desiccated material.     

Direct measurement of equilibrium constants for high-affinity hemoglobins

The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (K(D) < 1 micro M) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and approximately 100 micro M(-1) respectively, indicate that they are not capable of facilitating oxygen transport.     

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-[3H]piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. In contrast, the major differences between the kinetic binding parameters of agonists and antagonists to the low affinity agonist binding sites are in the association rate constants, which were 2-5 orders of magnitude lower for agonists. This demonstrates that there are basic differences in the interactions of agonists with the low and high affinity sites. Our findings also suggest that isomerization of the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.     

The extracellular-matrix-retaining cyanobacterium Nostoc verrucosum accumulates trehalose, but is sensitive to desiccation

The aquatic cyanobacterium Nostoc verrucosum forms macroscopic colonies, which consist of both cellular filaments and massive extracellular matrix material. In this study, the physiological features of N. verrucosum were investigated and compared with those of the anhydrobiotic cyanobacterium Nostoc commune. Nostoc verrucosum cells were sensitive to desiccation, but tolerant of freeze-thawing treatment in terms of both cell viability and photosynthetic O(2) evolution. Natural colonies of these cyanobacteria contained similar levels of chlorophyll a, carotenoids, the UV-absorbing pigments scytonemin and mycosporine-like amino acids, and uronic acid [a component of extracellular polysaccharides (EPS)]. EPS from both N. verrucosum and N. commune indicated low acidity and a high affinity for divalent cations, although their sugar compositions differed. The WspA protein, known to be a major component of the extracellular matrix of N. commune, was detected in N. verrucosum. Desiccation caused similarly high levels of trehalose accumulation in both cyanobacteria. Although previously considered relevant to anhydrobiosis in the terrestrial cyanobacterium N. commune, the data presented here suggest that extracellular matrix production and trehalose accumulation are not enough for standing extreme desiccation in N. verrucosum.     

Photomophogenesis in the Blue-green Alga Nostoc commune 584

The blue-green alga Nostoc commune 584 displays a photocontrolled developmental cycle similar to that described for N. muscorum A by Lazaroff and Vishniac (1961). In both species white fluorescent light acts at the same stage, ragulating the development of motile trichomes from sheathed aseriate colonies. However white light blocks this step in N. commune 584, whereas the formation of motile trichomes is promoted by white light in N. muscorum A. Light-grown (aseriate) cultures in N. commune 584 were used to determine the action spectra for photomorphogenesis. Green light (max 520 nm) inhbited aseriate colony breakage, and red light (max 640 nm) promoted colony breakage and the differentiation of motile trichomes. On a quantum basis green light was about 3 times more effective than red light. The morphogenetic effects of either red or green light were reversible by irradiation with the other color of light. Repeated photoreversibility was observed, and the algal culutres responded only to the color of the last irradiation in a sequence. An unidentified substance is excreted into the media of motile cultures of both N. commune 584 and N. muscorum A which promotes motility in non-motile cultures. The motility-promoting substances from both species are reciprocally active. Activity is lost when the media are autoclaved.     

Ligand binding properties of bacterial hemoglobins and flavohemoglobins

Bacterial hemoglobins and flavohemoglobins share a common globin fold but differ otherwise in structural and functional aspects. The bases of these differences were investigated through kinetic studies on oxygen, carbon monoxide, and nitric oxide binding. The novel bacterial hemoglobins from Clostridium perfringens and Campylobacter jejuni and the flavohemoglobins from Bacillus subtilis and Salmonella enterica serovar Typhi have been analyzed. Examination of the biochemical and ligand binding properties of these proteins shows a clear distinction between the two groups. Flavohemoglobins show a much greater tendency to autoxidation compared to bacterial hemoglobins. The differences in affinity for oxygen, carbon monoxide, and nitric oxide between bacterial hemoglobins and flavohemoglobins are mainly due to differences in the association rate constants. The second-order rate constants for oxygen and carbon monoxide binding to bacterial hemoglobins are severalfold higher than those for flavohemoglobins. A similar trend is observed for NO association with the oxidized iron(III) form of the proteins. No major differences are observed among the values obtained for the dissociation rate constants for the two groups of bacterial proteins studied, and these constants are all rather similar to those for myoglobin. Taken together, our data suggest that differences exist between the mechanisms of ligand binding to bacterial hemoglobins and flavohemoglobins, suggesting different functions in the cell.     

The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme

Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F-helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F-helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen-bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function.     

Kinetics and thermodynamics of oxygen, CO, and azide binding by the subcomponents of soybean leghemoglobin

Leghemoglobin shows extreme high affinity behavior in the binding of both oxygen and CO. We have determined the temperature dependence of the rate constants for ligation of oxygen and CO and from these data the thermodynamics (delta G0, delta H0, delta S0) of ligation for the purified components of soybean leghemoglobin. X-ray crystallography has shown that the heme cavity can easily accommodate ligands the size of nicotinate, and analysis of extended x-ray absorption fine structure data has shown that the Fe atom is in the mean plane of the heme in the leghemoglobin-CO complex. Ligation of oxygen and CO are in accord with this picture in that the Ea for oxygen binding is that expected for a diffusion controlled reaction and delta S0 for the ligation of both CO and oxygen is consistent with the simple immobilization of the ligand at the Fe, with no evidence for significant conformational changes in the protein or changes in solvation. At 20 degrees C the rate constants for oxygen and CO binding vary by 26-44% among the eight leghemoglobin components. For azide binding the variation is a factor of 2. These variations appear to arise from amino acid substitutions outside either the heme cavity or the two major paths for ligand entry to the heme. The distribution of leghemoglobin components varies with the age of the soybean nodule during the growing season. The changes in composition alone, however, would only allow the concentration of free oxygen to vary by about 3%. This finding calls into question models that ascribe a significant functional role to changes in the distribution of leghemoglobin components in regulating oxygen concentration in the nodule.     

The ultrastructure of immobilised desiccated cells of the cyanobacterium Nostoc commune UTEX 584

Cells of the cyanobacterium Nostoc commune UTEX 584 were immobilised, subjected to acute matric water stress (ψ_m = −128 MPa) and then desiccated. Their ultrastructure was investigated by the use of an anhydrous fixation procedure. Although shrunken and bleached, the integrity of the vegatative cells at the ultrastructural level was apparently preserved. The ease with which certain cyanobacterial cells can recover from desiccation may be consequent upon the maintenance of cellular organisation at the ultrastructural level.     

Binding of ferric heme by the recombinant globin from the cyanobacterium Synechocystis sp. PCC 6803

The product of the cyanobacterium Synechocystis sp. PCC 6803 gene slr2097 is a 123 amino acid polypeptide chain belonging to the truncated hemoglobin family. Recombinant, ferric heme-reconstituted Synechocystis sp. PCC 6803 hemoglobin is a low-spin complex whose endogenous hexacoordination gives rise to optical and NMR characteristics reminiscent of cytochrome b(5) [Scott, N. L., and Lecomte, J. T. J. (2000) Protein Sci. 9, 587-597]. In this work, the sequential assignments using (15)N-(13)C-labeled protein, (1)H nuclear Overhauser effects, and longitudinal relaxation data identified His70 as the proximal histidine and His46 as the sixth ligand to the iron ion. It was also found that one of two possible heme orientations within the protein matrix is highly preferred (>90%) and that this orientation is the same as in vertebrate myoglobins. The rate constant for the 180 degrees rotation of the heme within a protein cage to produce the favored isomer was 0.5 h(-1) at 25 degrees C, approximately 35 times faster than in sperm whale myoglobin. Variable temperature studies revealed an activation energy of 132 +/- 4 kJ mol(-1), similar to the value in metaquomyoglobin at the same pH. The rate constant for heme loss from the major isomer was estimated to be 0.01 h(-1) by optical spectroscopy, close to the value for myoglobin and decades slower than in the related Nostoc commune cyanoglobin. The slow heme loss was attributed in part to the additional coordination bond to His46, whereas the relatively fast rate of heme reorientation suggested that this bond was weaker than the proximal His70-Fe bond. The standard reduction potential of the hexacoordinated protein was measured with and without poly-L-lysine as a mediator and found to be approximately -150 mV vs SHE, indicating a stabilization of the ferric state compared to most hemoglobins and b(5) cytochromes.     

Ligand-induced conformation changes in Torpedo californica membrane-bound acetylcholine receptor

A time-dependent increase in ligand affinity has been studied in cholinergic ligand binding to Torpedocalifornica acetylcholine receptor by inhibition of the kinetics of of [125I]-alpha-bungarotoxin-receptor complex formation. The conversion of the acetylcholine receptor from low to high affinity form was induced by both agonists and antagonists of acetylcholine and was reversible upon removal of the ligand. The slow ligand induced affinity change in vitro resembled electrophysiological desensitization observed at the neuromuscular junction and described by a two-state model (Katz, B., & Thesleff, S. (1957) J. Physiol. 138, 63). A quantitative treatment of the rate and equilibrium constants determined for binding of the agonist carbamoylcholine to membrane bound acetylcholine receptor indicated that the two-state model is not compatible with the in vitro results.     

Molecular genetic and chemotaxonomic characterization of the terrestrial cyanobacterium Nostoc commune and its neighboring species

The phylogeny of the terrestrial cyanobacterium Nostoc commune and its neighboring Nostoc species was studied using molecular genetic and chemotaxonomic approaches. At least eight genotypes of N. commune were characterized by the differences among 16S rRNA gene sequences and the petH gene encoding ferredoxin-NADP? oxidoreductase and by random amplified polymorphic DNA analysis. The genotypes of N. commune were distributed in Japan without regional specificity. The nrtP gene encoding NrtP-type nitrate/nitrite permease was widely distributed in the genus Nostoc, suggesting that the occurrence of the nrtP gene can be one of the characteristic features that separate cyanobacteria into two groups. The wspA gene encoding a 36-kDa water stress protein was only found in N. commune and Nostoc verrucosum, suggesting that these Nostoc species that form massive colonies with extracellular polysaccharides can be exclusively characterized by the occurrence of the wspA gene. Fifteen species of Nostoc and Anabaena were investigated by comparing their carotenoid composition. Three groups with distinct patterns of carotenoids were related to the phylogenic tree constructed on the basis of 16S rRNA sequences. Nostoc commune and Nostoc punctiforme were clustered in one monophyletic group and characterized by the occurrence of nostoxanthin, canthaxanthin, and myxol glycosides.