Cyanide-linked dimer-monomer equilibrium of Chromatium vinosum ferric cytochrome c'.

Cyanide binding to Chromatium vinosum ferricytochrome c' has been studied to further investigate possible allosteric interactions between the subunits of this dimeric protein. Cyanide binding to C. vinosum cytochrome c' appears to be cooperative. However, the cyanide binding reaction is unusual in that the overall affinity of cyanide increases as the concentration of cytochrome c' decreases and that cyanide binding causes the ligated dimer to dissociate to monomers as shown by gel-filtration chromatography. Therefore, the cyanide binding properties of C. vinosum ferricytochrome c' are complicated by a cyanide-linked dimer to monomer dissociation equilibrium of the complexed protein. The dimer to monomer dissociation constant is 20-fold smaller than that for CO linked dissociation constant of ferrocytochrome c'. Furthermore, the pH dependence of both the intrinsic equilibrium binding constant and the dimer to monomer equilibrium dissociation constant was investigated over the pH range of 7.0 to 9.2 to examine the effect of any ionizable groups. The equilibrium constants did not exhibit a significant pH dependence over this pH range.

Crystallization and characterization of Chromatium vinosum cytochrome c'.

The dimeric high spin c-type cytochrome c' from Chromatium vinosum has been crystallized and the crystals characterized by x-ray diffraction. This cytochrome c' exhibits ligand-controlled dissociation from a dimer to a monomer upon binding carbon monoxide and represents an opportunity to obtain unique information concerning cooperativity in heme proteins. The C. vinosum cytochrome c' protein crystals are grown from polyethylene glycol 4000 and grow in both space group P2(1)2(1)2(1) (a = 49.2, b = 56.7, c = 98.8 A) and space group P2(1) (a = 55, b = 94, c = 50, beta = 106.1 A) depending upon the growth rate, with the P2(1)2(1)2(1) form favored at slower growth rates. The high resolution (2.0 A) atomic structure of the P2(1)2(1)2(1) form is being determined.

1H NMR characterization of Chromatium gracile high-potential iron protein and its ruthenium-modified derivatives. Modulation of the reduction potentials in low- and high-potential [Fe4S4] ferredoxins.

The NMR spectra of the high-potential iron protein from the photosynthetic bacterium Chromatium gracile and its ruthenium-labeled (His-42 and His-20) derivatives are reported. The isotropically shifted resonances in both the oxidized and reduced forms show a complex pH dependence due to the presence of three ionizable residues (Glu-44, His-20, and His-42). Assignments have been made to specific residues and the spectral features compared to those of other bacterial HiPIP's. The decrease in the reduction potential with increasing pH for this class of proteins is attributed to stabilization of the oxidized state of the cluster by delocalization of electron density onto the neighboring Tyr-19 residue.

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.

Thioredoxin from Rhodospirillum rubrum: primary structure and relation to thioredoxins from other photosynthetic bacteria.

Thioredoxin was isolated from a photosynthetic purple nonsulfur bacterium, Rhodospirillum rubrum, and its primary structure was determined by high-performance tandem mass spectrometry. The sequence identity of R. rubrum thioredoxin to Escherichia coli thioredoxin was intermediate to those of the Chlorobium thiosulfatophilum and Chromatium vinosum proteins. The results indicate that R. rubrum has an NADP-thioredoxin system similar to that of other photosynthetic purple bacteria.

Spectroscopic and ligand-binding properties of an oxygen-binding heme protein from Chromatium vinosum.

Magnetic circular dichroism spectra were obtained for the oxidized and reduced forms of cyanide, azide and carbon monoxide complexes of an O2-binding hemeprotein isolated from the photosynthetic purple sulfur bacterium, Chronatium vinosum. Cyanide binding to the protein, which results in formation of a low-spin complex, was highly pH dependent with little complex formation observed at pH values near or below 7.

[Detection by the use of silver ions of additional protein zones in gradient polyacrylamide gels stained with Coomassie]. / Vyiavlenie s pomoshch'iu ionov serebra dopolnitel'nykh zon belkov v gradientnykh poliakrilamidnykh geliakh, okrashennykh kumassi.

A simple method of revealing the additional zones of proteins in gradient polyacrylamide gels, preliminary dyed Coomassie by means of silver ions is described. The dyeing of Coomassie allows to avoid the time-consuming stages of preliminary treatment of gels as well to reveal more sensitive zones in gels. On the second stage of dyeing silver minor zones appear there which were not seen while Coomassie was dyed. The suggested method preserves high sensitivity characteristic of the methods of gel dyeing with silver.

Properties of the reaction center of the thermophilic purple photosynthetic bacterium Chromatium tepidum.

Reaction centers were purified from the thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. The reaction center consists of four polypeptides L, M, H and C, whose apparent molecular masses were determined to be 25, 30, 34 and 44 kDa, respectively, by polyacrylamide gel electrophoresis. The heaviest peptide corresponds to tightly bound cytochrome. The tightly bound cytochrome c contains two types of heme, high-potential c-556 and low-potential c-553. The low-potential heme is able to be photooxidized at 77 K. The reaction center exhibits laser-flash-induced absorption changes and circular dichroism spectra similar to those observed in other purple photosynthetic bacteria. Whole cells contain both ubiquinone and menaquinone. Reaction centers contain only a single active quinone; chemical analysis showed this to be menaquinone. Reaction center complexes without the tightly bound cytochrome were also prepared. The near-infrared pigment absorption bands are red-shifted in reaction centers with cytochrome compared to those without cytochrome.

The primary structure of thioredoxin from Chromatium vinosum determined by high-performance tandem mass spectrometry.

The primary structure of thioredoxin, a redox protein isolated from Chromatium vinosum, was determined by high-performance tandem mass spectrometry, which permitted sequencing of the 14 peptides (ranging in length from 2 to 18 amino acids) generated by digestion with trypsin and of several peptides produced by Staphylococcus aureus protease. The mass spectrometrically determined molecular weights of the peptides from the latter digest were used to properly align the tryptic peptides, which could also be accomplished on the basis of the considerable homology with Escherichia coli thioredoxin. Finally, the molecular weight of the Chromatium thioredoxin was determined by mass spectrometry and found to be 11,748.0, in good agreement with 11,750.2 calculated for the proposed sequence. Although it was difficult to establish by mass spectrometry, five leucines and three isoleucines could be identified, leaving only eight undifferentiated.

Spectroscopic and kinetic properties of an oxygen-binding heme protein from Chromatium vinosum.

Resonance Raman and electron paramagnetic resonance spectroscopy have been utilized to identify histidine as an axial heme ligand in a high spin, heme c-containing protein isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum. Resonance Raman spectroscopy has also been used to characterize the CO adduct of the C. vinosum hemoprotein. Resonance Raman spectra of the heme site obtained within 10 ns of CO photolysis from the ferrous hemoprotein are virtually identical to those of the unligated protein, indicating that there is little or no rearrangement of the heme pocket in response to ligand photolysis. The equilibrium constant for CO binding to the ferrous hemeprotein was measured to be 1.7 X 10(-5) M-1 and the CO association rate constant determined to be 5.4 X 10(3) M-1 S-1. The quantum efficiency for photodissociation of the hemoprotein X CO complex was greater than or equal to 0.9.

Isolation, characterization, and comparison of a ubiquitous pigment-protein complex consisting of a reaction center and light-harvesting bacteriochlorophyll proteins present in purple photosynthetic bacteria.

Protein complexes (photochemical reaction complex; PR complex) bound to both light-harvesting bacteriochlorophyll-1 (LH-Bchl-1) and reaction center Bchl (RC-Bchl) were purified from Rhodospirillum rubrum (wild and carotenoid-less), Rhodopseudomonas sphaeroides (wild), and Chromatium vinosum (wild). Another protein complex (LH-2 complex) bound to LH-Bchl-2 was also purified from Rps. sphaeroides. The bacteria were grown in the presence of a [14C]amino acid mixture. The purification procedure included molecular-sieve chromatography in the presence of cholate-deoxycholate, and non-equilibrated isoelectric electrophoresis with 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate. The purified complexes were separated into their constituent proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The molar ratios of the proteins were determined by comparing their radioactivities divided by their molecular weights after consideration of the molecular masses of the complexes. The PR complexes all contained per mol: 1 mol each of RC H-, M-, and L-subunits, 10-13 (probably 12) mol each of two other proteins with molecular weights of 11-12K and 8-11K, 28-32 mol Bchl, 13-15 mol carotenoids (except in the carotenoid-less mutant), 2.6-3.9 mol ubiquinone (or menaquinone in Chr. vinosum), and 53-79 mol phosphate without phospholipid. The LH-2 complex contained per mol: 1 mol 52K protein, about 13 (probably 12) mol each of 9K and 8K proteins, 30 mol Bchl, 10 mol carotenoids, and 38 mol phosphate without phospholipid. The PR complexes and LH-2 complex showed similar X-ray diffraction patterns, implying that they had similar, highly organized molecular structures.

Resonance Raman characterization of a novel, oxygen-binding heme protein from Chromatium vinosum.

Resonance Raman spectroscopy was employed to characterize the local heme environment of a high-spin, ligand-binding heme protein from Chromatium vinosum (Chromatium high-spin hemoprotein). High-frequency spectra obtained with both B- and Q-band excitation were found to resemble qualitatively those of deoxyhemoglobin (HbA). Differences between HbA and Chromatium high-spin hemoprotein spectra can be assigned to either the effects of a covalent linkage of the heme vinyls to the protein matrix or alterations in the heme-proximal ligand bonding interaction. Both kinematic and electronic effects were evident. The behavior of heme core-size sensitive modes and low-frequency modes in Chromatium high-spin hemoprotein may be an indication of distortions in the heme geometry of Chromatium high-spin hemoprotein relative to HbA. The effects of covalent bonding of the heme peripheral vinyls upon the vibrational, electronic, and geometric characteristics of the heme active site in Chromatium high-spin hemoprotein are discussed.

Terminal steps of bacteriochlorophyll a phytol formation in purple photosynthetic bacteria.

Four chemically different bacteriochlorophylls (Bchls) a esterified with geranylgeraniol, dihydrogeranylgeraniol, tetrahydrogeranylgeraniol, and phytol have been detected by high-pressure liquid chromatography in cell extracts from Rhodopseudomonas sphaeroides and Chromatium vinosum. Bchl a containing phytol is the principal component, and the other three Bchls a comprise about 4% of the total Bchls a in stationary-phase cells of R. sphaeroides and C. vinosum. The high levels of the minor pigments occur in the beginning of Bchl a phytol formation, indicating that they are not degradation products, but intermediates of Bchl a phytol formation.

Cytochromes and anaerobic sulfide oxidation in the purple sulfur bacterium Chromatium warmingii.

Two soluble acidic c-type cytochromes--c' and c-552--were isolated by ion exchange chromatography, gel filtration and ammonium sulfate fractionation. Cytochrome c' is a high-spin cytochrome with maxima at 399 nm, 490 nm, and 634 nm in the oxidized form and at 550 nm, 425 nm and a characteristic shoulder at 434 nm in the reduced state. The best purity index obtained (A280/A399) was 0.35. Cytochrome c' is autoxidizable, has a molecular weight of 12000 (estimated by sodium dodecylsulfate electrophoresis), a midpoint redoxpotential of +10 mV and an isoelectric point at pH 4.0. The reduced cytochrome c' reacts with carbon monoxide. The reaction is reversible. Cytochrome c-552 shows maxima at 552 nm, 523 nm and 417 nm in the reduced form and at 408 nm in the oxidized state. The best purity index obtained (A280/A408) was 0.94. Cytochrome c-552 has a molecular weight of 30000 and an isoelectric point between pH 4.3 and 5.0. Chromatium warmingii also contains a membrane-bound cytochrome c-552. During anaerobic sulfide oxidation, elemental sulfur and sulfate were formed at the same time. When all sulfide was consumed by the cells, the remaining intracellular elemental sulfur was further oxidized to sulfate.

Molecular properties of high potential iron sulfur protein of Chromatium warmingii.

High potential iron sulfur protein (HIPIP) of the purple sulfur bacterium Chromatium warmingii was purified to homogeneity by ion exchange chromatography, gel filtration and ammonium sulfate fractionation. The acidic protein was isolated in the reduced form. The best purity index (A280/A388) obtained was 2.52, and 3.8 mumol of the protein was isolated out of 100 g wet cell material. The molecular weights estimated by sodium dodecylsulfate polyacrylamide gel electrophoresis and gel filtration through Sephacryl S-200 were 8900 and 10 500, respectively. The protein has an isoelectric point at pH 3.6 for the reduced form and at pH 3.8 for the oxidized form, and a midpoint redox potential of +355 mV. One mol of HIPIP contains 4 mol nonheme iron and 4 mol acid-labile sulfur.

Partial purification and characterization of two soluble c-type cytochromes from Chromatium vinosum.

Two c-type cytochromes from Chromatium vinosum have been partially purified and characterized. Cytochrome c550, which appears to function as an electron carrier in the cyclic electron transport chain of this photosynthetic purple sulfur bacterium, has a molecular weight of approximately 15,000 and an oxidation-reduction midpoint potential (Em) of +240 mV at pH 7.4. It has (in the reduced form) an alpha band at 550 nm and a beta band at 520 nm. Cytochrome c551 is characterized by absorbance maxima at 354 and 409 nm in the oxidized form and 418, 523, and 551 nm in the reduced form. The reduced cytochrome reacts with CO. Cytochrome c551 is a monomeric protein with a molecular weight of 18,800 +/- 700 and Em = -299 +/- 5 mV (pH independent between pH 6.3 and 8.0). It appears to lack a methionine axial ligand as indicated by the absence of an absorbance band at 695 nm in the oxidized form.

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.