Amino Acid Composition and Terminal Sequences of Ferredoxins from Two Photosynthetic Green Bacteria

The amino acid composition of ferredoxins from Chlorobium thiosulfatophilum 8327 and Chloropseudomonas ethylicum, like C. thiosulfatophilum Tassajara, resembled ferredoxins from nonphotosynthetic anaerobes rather than Chromatium; the terminal sequences, however, more closely resembled Chromatium ferredoxin.     

Efficiency of ferredoxins and flavodoxins as mediators in systems for hydrogen evolution.

1. The efficiencies of ferredoxins and flavodoxins from a range of sources as mediators in systems for hydrogen evolution were assessed. 2. In supporting electron transfer from dithionite to hydrogenase of the bacterium Clostridium pasteurianum, highest activity was shown by the ferredoxin from the cyanobacterium Chlorogloeopsis fritschii and flavodoxin from the bacterium Megasphaera elsdenii. The latter was some twenty times as active as comparable concentrations of Methyl Viologen. Ferredoxins from the cyanobacterium Anacystis nidulans and the red alga Porphyra umbilicalis also showed high activity. 3. In mediating electron transfer from chloroplast membranes to Clostridium pasteurianum hydrogenase the flavodoxin from Anacystis nidulans proved the most active with Nostoc strain MAC flavodoxin and Porphyra umbilicalis ferredoxin also being appreciably more active than other cyanobacterial and higher plant ferredoxins. 4. In both hydrogenase systems the ferredoxin and flavodoxin from the red alga Chondrus crispus and the ferredoxin from another red alga Gigartina stellata showed very low activity. 5. There appeared to be no apparent correlation of efficiency in supporting hydrogenase activity with midpoint redox potential (Em) of the mediators, though some correlation of Em with the efficiency of the mediators in supporting NADP+ photoreduction by chloroplasts, or pyruvate oxidation by a Clostridium pasteurianum system, was evident. 6. Activity of the mediators in the hydrogenase systems therefore primarily reflects differences in tertiary structure conferring differing affinities for the other components of the systems.     

Unusual spectroscopic and electrochemical properties of the 2[4Fe-4S] ferredoxin of Thauera aromatica

A reduced ferredoxin serves as the natural electron donor for key enzymes of the anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica. It contains two [4Fe-4S] clusters and belongs to the Chromatium vinosum type of ferredoxins (CvFd) which differ from the "clostridial" type by a six-amino acid insertion between two successive cysteines and a C-terminal alpha-helical amino acid extension. The electrochemical and electron paramagnetic resonance (EPR) spectroscopic properties of both [4Fe-4S] clusters from T. aromatica ferredoxin have been investigated using cyclic voltammetry and multifrequency EPR. Results obtained from cyclic voltammetry revealed the presence of two redox transitions at -431 and -587 mV versus SHE. X-band EPR spectra recorded at potentials where only one cluster was reduced (greater than -500 mV) indicated the presence of a spin mixture of S = (3)/(2) and (5)/(2) spin states of one reduced [4Fe-4S] cluster. No typical S = (1)/(2) EPR signals were observed. At lower potentials (less than -500 mV), the more negative [4Fe-4S] cluster displayed Q-, X-, and S-band EPR spectra at 20 K which were typical of a single S = (1)/(2) low-spin [4Fe-4S] cluster with a g(av) of 1.94. However, when the temperature was decreased stepwise to 4 K, a magnetic interaction between the two clusters gradually became observable as a temperature-dependent splitting of both the S = (1)/(2) and S = (5)/(2) EPR signals. At potentials where both clusters were reduced, additional low-field EPR signals were observed which can only be assigned to spin states with spins of >(5)/(2). The results that were obtained establish that the common typical amino acid sequence features of CvFd-type ferredoxins determine the unusual electrochemical properties of the [4Fe-4S] clusters. The observation of different spin states in T. aromatica ferredoxin is novel among CvFd-type ferredoxins.     

Thioredoxin system of the photosynthetic anaerobe Chromatium vinosum

Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles aerobic bacterial cells in that it has an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. Both protein components were purified to homogeneity, and some of their properties were determined. Chromatium vinosum thioredoxin was slightly larger than other bacterial thioredoxins (13 versus 12 kilodaltons) but was similar in its specificity (ability to activate chloroplast NADP-malate dehydrogenase more effectively than chloroplast fructose-1,6-bisphosphatase) and immunological properties. As in other bacteria, Chromatium vinosum NADP-thioredoxin reductase was an arsenite-sensitive flavoprotein composed of two 33.5-kilodalton subunits, that required thioredoxin for the NADPH-linked reduction of 5,5'-dithiobis(2-nitrobenzoic acid). Chromatium vinosum NADP-thioredoxin reductase very effectively reduced several different bacterial-type thioredoxins (Escherichia coli, Chlorobium thiosulfatophilum (this name has not been approved by the International Committee of Systematic Bacteriology), Rhizobium meliloti) but not others (Clostridium pasteurianum, spinach chloroplast thioredoxin m). The results show that Chromatium vinosum contains an NADP-thioredoxin system typical of evolutionarily more advanced microorganisms.     

Heterologous hybridization of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) restores the enzyme activities

The catalytic core (A8) and small subunit (B) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were isolated from two species of cyanobacteria (Aphanothece halophytica and Synechococcus ACMM 323) as well as from the photosynthetic purple sulfur bacterium, Chromatium vinosum. The subunit B is essential for the activity of all three enzymes. The heterologous hybridization of RuBisCO molecules from the three organisms was attempted and the reconstitution of the catalytically active hybrid was achieved between A8 derived from either Aphanothece or Synechococcus and subunit B from Aphanothece, Synechococcus or Chromatium. However, reconstitution of the enzymically active hybrid between A8 from Chromatium and B subunits from the cyanobacteria could not be achieved. Experiments by using high performance liquid column chromatography also showed the formation of a heterologous hybrid possessing RuBP carboxylase activity.     

Expression of genes for subunits of plant-type RuBisCO from Chromatium and production of the enzymically active molecule in Escherichia coli

A DNA fragment containing genes for both large (A) and small (B) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from a photosynthetic bacterium Chromatium vinosum was ligated with vectors for expressing unfused proteins and introduced into cells of Escherichia coli. The expressers of RuBisCO were screened on agar plates using the specific antibody raised against the native enzyme from Chromatium. The production of both subunits A and B in the expressers was demonstrated by an immunoblotting experiment. The amount of RuBisCO produced in the E. coli cells was as high as 15% of the total soluble protein after induction with isopropyl-beta-D-thiogalactoside. The specific activity of enzyme molecules produced in E. coli was nearly the same as that of the original Chromatium enzyme. On gel filtration high-performance liquid chromatography the two enzymes showed identical elution behavior, strongly indicating their similar quaternary structures.     

The iron electron-nuclear double resonance (ENDOR) of 4-Fe clusters in iron-sulfur proteins from Chromatium and Clostridium pasteurianum.

Iron electron-nuclear double resonance (ENDOR) measurements were made of the 4-Fe clusters in oxidized Chromatium high-potential iron-sulfur protein, dithionite-reduced high-potential iron-sulfur protein in 80% dimethylsulphoxide, fully reduced Clostridium pasteurianum ferredoxin in aqueous solution and in 80% dimethylsulfoxide. The hyperfine couplings determined show that: i) the electron distribution in each case is nearly symmetric; ii) there are two types of iron in oxidized high potential iron-sulfur protein; iii) only one type of iron is observed in each fully reduced 4-Fe cluster; iv) the data also suggest a greater electron delocalization onto the ligands as compared to the 2-Fe ferredoxins.     

Chromatium glycolicum sp. nov., a moderately halophilic purple sulfur bacterium that uses glycolate as substrate

From the microbial mats that develop in Solar Lake, a new purple sulfur bacterium was isolated. This strain (Chromatium strain SL 3201) was morphologically similar to Chromatium gracile and Chromatium minutissimum. Chromatium SL 3201 was found to be a moderate halophile with a growth range between 2 and 20% NaCl (optimum 4–5% NaCl) and was able to grow photo-organotrophically using glycolate and glycerol. It is the first described phototrophic sulfur bacterium able to use glycolate. According to NaCl requirements and utilization of organic compounds, the strain is not related to any known species of the genus Chromatium. On the basis of its 16S rRNA gene sequence, it clusters with other Chromatium species and is most similar to Chromatium salexigens and Chr. gracile, but it is sufficiently separated to be considered as a new species of the genus. It is, therefore, described as Chromatium glycolicum sp. nov. Received: 17 June 1996 / Accepted: 4 November 1996     

Comparison of the properties of two hydrogenases from the photosynthetic bacterium Chromatium vinosum

Chromatium vinosum hydrogenases I and II were purified to specific activities of 9.6 and 28.0 units/mg protein, respectively. They have the same isoelectric point (pI = 4.1), and their visible spectra are typical of iron-sulfur proteins. Hydrogenase II in general was more stable than hydrogenase I. Both enzymes lost their activities slowly during storage in air, and this inactivation was more apparent in preparations of hydrogenase I. Bovine serum albumin helped to stabilize hydrogenase I against thermal and storage inactivation. The pH optima of H2-evolution activity of hydrogenases I and II were 7.4 and 5.4, respectively. Neither enzyme was able to evolve H2 from reduced ferredoxins as the sole electron carrier, but ferredoxins had an effect on the activity with methyl viologen as carrier to hydrogenase I. None of the natural compounds tested was able to serve as a physiological donor for H2 production. Hydrogenase I was more susceptible than hydrogenase II to inhibition by heavy metal ions and other enzyme inhibitors. Both enzymes were reversibly inhibited by CO with Ki values of 12 and 6 Torr for hydrogenase I and II, respectively. Hydrogenase I was more sensitive to denaturation by urea and guanidinium chloride while hydrogenase II was more susceptible to sodium dodecyl sulfate. Both enzymes were rapidly and irreversibly inactivated by dimethyl sulfoxide. Hydrogenase I evolved H2 from methyl viologen and ferredoxin photoreduced by chloroplasts. The enzymes differed in their iron and acid-labile sulfur contents.     

Crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum: evolutionary and mechanistic inferences for [3/4Fe-4S] ferredoxins.

The crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum has been solved by molecular replacement using data recorded with synchrotron radiation. The crystals were hexagonal prisms that showed a strong tendency to develop into long tubes. The hexagonal prisms diffracted to 2.1 A resolution at best, and a structural model for C. vinosum ferredoxin has been built with a final R of 19.2%. The N-terminal domain coordinates the two [4Fe-4S] clusters in a fold that is almost identical to that of other known ferredoxins. However, the structure has two unique features. One is a six-residue insertion between two ligands of one cluster forming a two-turn external loop; this short loop changes the conformation of the Cys 40 ligand compared to other ferredoxins and hampers the building of one NH...S H-bond to one of the inorganic sulfurs. The other remarkable structural element is a 3.5-turn alpha-helix at the C-terminus that covers one side of the same cluster and is linked to the cluster-binding domain by a six-residue external chain segment. The charge distribution is highly asymmetric over the molecule. The structure of C. vinosum ferredoxin strongly suggests divergent evolution for bacterial [3/4Fe-4S] ferredoxins from a common ancestral cluster-binding core. The unexpected slow intramolecular electron transfer rate between the clusters in C. vinosum ferredoxin, compared to other similar proteins, may be attributed to the unusual electronic properties of one of the clusters arising from localized changes in its vicinity rather than to a global structural rearrangement.     

Carbon isotope fractionation by thermophilic phototrophic sulfur bacteria: evidence for autotrophic growth in natural populations.

Purple phototrophic bacteria of the genus Chromatium can grow as either photoautotrophs or photoheterotrophs. To determine the growth mode of the thermophilic Chromatium species, Chromatium tepidum, under in situ conditions, we have examined the carbon isotope fractionation patterns in laboratory cultures of this organism and in mats of C. tepidum which develop in sulfide thermal springs in Yellowstone National Park. Isotopic analysis (13C/12C) of total carbon, carotenoid pigments, and bacteriochlorophyll from photoautotrophically grown cultures of C. tepidum yielded 13C fractionation factors near -20%. Cells of C. tepidum grown on excess acetate, wherein synthesis of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase ribulose bisphosphate carboxylase) was greatly repressed, were isotopically heavier, fractionation factors of ca. -7% being observed. Fractionation factors determined by isotopic analyses of cells and pigment fractions of natural populations of C. tepidum growing in three different sulfide thermal springs in Yellowstone National Park were approximately -20%, indicating that this purple sulfur bacterium grows as a photoautotroph in nature.     

Carbon isotope fractionation by thermophilic phototrophic sulfur bacteria: evidence for autotrophic growth in natural populations

Purple phototrophic bacteria of the genus Chromatium can grow as either photoautotrophs or photoheterotrophs. To determine the growth mode of the thermophilic Chromatium species, Chromatium tepidum, under in situ conditions, we have examined the carbon isotope fractionation patterns in laboratory cultures of this organism and in mats of C. tepidum which develop in sulfide thermal springs in Yellowstone National Park. Isotopic analysis (13C/12C) of total carbon, carotenoid pigments, and bacteriochlorophyll from photoautotrophically grown cultures of C. tepidum yielded 13C fractionation factors near -20%. Cells of C. tepidum grown on excess acetate, wherein synthesis of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase ribulose bisphosphate carboxylase) was greatly repressed, were isotopically heavier, fractionation factors of ca. -7% being observed. Fractionation factors determined by isotopic analyses of cells and pigment fractions of natural populations of C. tepidum growing in three different sulfide thermal springs in Yellowstone National Park were approximately -20%, indicating that this purple sulfur bacterium grows as a photoautotroph in nature.     

Two distinct ferredoxins from Rhodobacter capsulatus: complete amino acid sequences and molecular evolution

Two distinct ferredoxins were purified from Rhodobacter capsulatus SB1003. Their complete amino acid sequences were determined by a combination of protease digestion, BrCN cleavage and Edman degradation. Ferredoxins I and II were composed of 64 and 111 amino acids, respectively, with molecular weights of 6,728 and 12,549 excluding iron and sulfur atoms. Both contained two Cys clusters in their amino acid sequences. The first cluster of ferredoxin I and the second cluster of ferredoxin II had a sequence, CxxCxxCxxxCP, in common with the ferredoxins found in Clostridia. The second cluster of ferredoxin I had a sequence, CxxCxxxxxxxxCxxxCM, with extra amino acids between the second and third Cys, which has been reported for other photosynthetic bacterial ferredoxins and putative ferredoxins (nif-gene products) from nitrogen-fixing bacteria, and with a unique occurrence of Met. The first cluster of ferredoxin II had a CxxCxxxxCxxxCP sequence, with two additional amino acids between the second and third Cys, a characteristics feature of Azotobacter-[3Fe-4S] [4Fe-4S]-ferredoxin. Ferredoxin II was also similar to Azotobacter-type ferredoxins with an extended carboxyl (C-) terminal sequence compared to the common Clostridium-type. The evolutionary relationship of the two together with a putative one recently found to be encoded in nifENXQ region in this bacterium [Moreno-Vivian et al. (1989) J. Bacteriol. 171, 2591-2598] is discussed.     

Polar Lipids of Chromatium Strain D Grown at Different Light Intensities

The total amount and the composition of polar lipids of the photosynthetic bacterium Chromatium strain D grown in high (7,500 ft/c) and low (100 ft/c) incident white light were measured. Cells grown with low incident light as compared with cells grown in high incident light contained approximately 3.5 times more chlorophyll and contained significantly more internal membranes (chromatophores) as determined by electron microscopy. Nevertheless, there was approximately 40% more phospholipid in cells grown at 7,500 ft/c than in cells grown with 100 ft/c; the phospholipid composition of the cells was similar under both conditions of growth. These observations lead us to suggest that the organization and perhaps localization of the phospholipids in Chromatium may be dependent on the light intensity supplied during growth.     

Purification and properties of two ferredoxins from the nitrogen-fixing bacterium Bacillus polymyxa

Two ferredoxins, designated FdI and FdII, have been isolated from the nitrogen-fixing bacterium Bacillus polymyxa. The two ferredoxins were readily separated on DEAE-cellulose and disc gel electrophoresis. The amino acid compositions of FdI and FdII showed them to be different protein species; the greater number of acidic amino acid residues in FdI than in FdII appears to account for separation based on electronic charge. FdI and FdII were both found to have four nonheme iron and four acid-labile sulfur groups per mole. The absorption spectra of the two ferredoxins are almost identical, with a peak in the visible region of the spectrum at 385 nm. The $\text{A}{}_{358}\text{A}{}_{280}$ absorbance ratio of both ferredoxins was 0.54–0.55. FdII was not stable under aerobic conditions, as indicated by a decrease in the visible region of the spectrum. Both FdI and FdII have nearly identical molecular weights, as judged by gel filtration and amino acid composition (approx 8800).The purified ferredoxins catalyzed the photoreduction of NADP by spinach chloroplasts with equal effectiveness. In the nitrogen-fixation reaction of B. polymyxa, FdII was more effective than FdI.     

Composition of the Sulfur Particle of Chromatium vinosum Strain D

Sulfur particles were isolated from the purple sulfur photosynthetic bacterium, Chromatium vinosum strain D. The composition of these particles was determined to be 93% sulfur, 5% protein, and 0.6% lipid. Gel electrophoresis indicated the presence of a single protein species with a molecular weight of 13,500 daltons. From these results, the sulfur particle is postulated to be bounded by a membrane consisting entirely of protein.     

Azotobacter vinelandii ferredoxin I: a sequence and structure comparison approach to alteration of [4Fe-4S]2+/+ reduction potential.

The reduction potential (E(0)') of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (AvFdI) and related ferredoxins is approximately 200 mV more negative than the corresponding clusters of Peptostreptococcus asaccharolyticus ferredoxin and related ferredoxins. Previous studies have shown that these differences in E(0)' do not result from the presence or absence of negatively charged surface residues or in differences in the types of hydrophobic residues found close to the [4Fe-4S](2+/+) clusters. Recently, a third, quite distinct class of ferredoxins (represented by the structurally characterized Chromatium vinosum ferredoxin) was shown to have a [4Fe-4S](2+/+) cluster with a very negative E(0)' similar to that of AvFdI. The observation that the sequences and structures surrounding the very negative E(0)' clusters in quite dissimilar proteins were almost identical inspired the construction of three additional mutations in the region of the [4Fe-4S](2+/+) cluster of AvFdI. The three mutations, V19E, P47S, and L44S, that incorporated residues found in the higher E(0)' P. asaccharolyticus ferredoxin all led to increases in E(0)' for a total of 130 mV with a 94-mV increase in the case of L44S. The results are interpreted in terms of x-ray structures of the FdI variants and show that the major determinant for the large increase in L44S is the introduction of an OH-S bond between the introduced Ser side chain and the Sgamma atom of Cys ligand 42 and an accompanying movement of water.     

Lipopolysaccharides of Thiocystis violacea, Thiocapsa pfennigii, and Chromatium tepidum, species of the family Chromatiaceae.

The lipopolysaccharides (LPS) of three species of purple sulfur bacteria (Chromatiaceae), Thiocystis violacea, Thiocapsa pfennigii, and the moderately thermophilic bacterium Chromatium tepidum, were isolated. The LPS of Thiocystis violacea and Chromatium tepidum contained typical O-specific sugars, indicating O-chains. Long O-chains were confirmed for these species by sodium deoxycholate gel electrophoresis of their LPS. Thiocapsa pfennigii, however, had short or no O-chains. The core region of the LPS of all three species comprised D-glycero-D-mannoheptose as the only heptose and 2-keto-3-deoxyoctonate. The lipid A, obtained from the LPS by mild acid hydrolysis, contained glucosamine as the main amino sugar. Amide-bound 3-hydroxymyristic acid was the only hydroxy fatty acid. The main ester-bound fatty acid in all lipid A fractions was 12:0. Mannose and small amounts of 2,3-diamino-2,3-dideoxy-D-glucose were common constituents of the lipid A of the three Chromatiaceae species investigated. All lipid A fractions were essentially free of phosphate.