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PubMed1996

Evaluation of Solvent Accessibility to the [Fe(4)S(4)] Binding Pocket in Native and Tyr19 Mutant High Potential Iron Proteins by (1)H-(15)N HMQC and (19)F NMR Experiments.

The solvent accessibility of Chromatium vinosumhigh potential iron protein (HiPIP) has been investigated by use of (1)H-(15)N HMQC, and (19)F NMR spectroscopy. These NMR experiments indicate that solvent accessibility to the cluster core is similar, and minimal, for the reduced and oxidized states of native HiPIP, but increases significantly for mutant proteins (Tyr19Leu and Tyr19His). These results support a proposed role [Agarwal, A.; Li, D.; Cowan, J. A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 9440-9444] for Tyr19 in maintaining hydrolytic stability of the [Fe(4)S(4)] cluster, and demonstrate a general strategy for mapping out the solvent accessibility of protein-bound metalloredox prosthetic centers.

Journal Article↗
PubMed2002

Genetic construction of truncated and chimeric metalloproteins derived from the alpha subunit of acetyl-CoA synthase from Clostridium thermoaceticum.

In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha(2)beta(2) tetrameric 310 kDa bifunctional enzyme. ACS catalyzes the reversible reduction of CO(2) to CO and the synthesis of acetyl-CoA from CO (or CO(2) in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains seven metal-sulfur clusters of four different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kDa beta subunit, while the A-cluster, a Ni-X-Fe(4)S(4) cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kDa alpha subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire beta subunit was removed, as long as CO, rather than CO(2) and a low-potential reductant, was used as a substrate. Truncating an approximately 30 kDa region from the N-terminus of the alpha subunit yielded a 49 kDa protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO-binding properties. Further truncation afforded a 23 kDa protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of [Fe(4)S(4)](2+) clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 and 82 kDa fragments of the alpha subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multicentered metalloenzymes and to generate new complex metalloenzymes with interesting properties.

Acetate-CoA Ligase↗
PubMed2000

Characterization of the cys gene locus from Allochromatium vinosum indicates an unusual sulfate assimilation pathway.

Homologues of the genes cysB, cysI, cysH, cysD, cysN, and selD were identified in the genome of the phototrophic purple sulfur bacterium Allochromatium vinosum (formerly Chromatium vinosum). On the basis of amino acid comparisons these genes encode a ferredoxin-dependent siroheme-sulfite reductase (CysI), a plant-type assimilatory APS reductase without thioredoxin domain (CysH), the two different subunits of heterodimeric ATP sulfurylase (CysDN), a transcriptional regulator (CysB) and a selenophosphate synthase (SelD). cysIHDN appear to form an operon and are preceded by cysB which is transcribed in the opposite direction. SelD is situated downstream of cysN and transcribed divergently to cysIHDN. The lack of a gene for APS kinase and presence of a gene for an assimilatory APS reductase implies that assimilatory sulfate reduction in A. vinosum proceeds along the pathway suggested for higher plants without intermediary formation of PAPS. Two completely separate pathways involving specialized enzymes are used for assimilatory sulfate reduction and dissimilatory sulfur oxidation in A. vinosum. The presence of cysB indicates that the genes for assimilatory sulfate reduction are expressed only in the absence of reduced sulfur compounds.

Amino Acid Sequence↗
PubMed2002

Transcription of three sets of genes coding for the core light-harvesting proteins in the purple sulfur bacterium, Allochromatium vinosum.

The nucleotide sequence of the puf operon coding for the subunits of the photosynthetic reaction center and the core light-harvesting complex (LH1) of the purple sulfur bacterium, Allochromatium (A.) vinosum (formally Chromatium vinosum), was completely determined. Unlike other known puf operons, which contain only one set of genes coding for the LH1 apoproteins, pufB and pufA, the A. vinosum puf operon included three sets of pufB and pufA genes with a gene order of pufB (1) A (1) LMCB (2) A (2) B (3) A (3). Northern hybridization analysis suggested that all of the nine puf genes are co-transcribed as a 4.43 kb mRNA. Three small mRNAs corresponding to pufB (2) A (2) B (3) A (3), pufB (2) A (2) B (3), and pufB (2) A (2) were detected, as well as two small mRNAs covering pufB (1) A (1). Analysis of the nucleotide sequence of the puf operon, including the flanking regions and 5'-ends of the six mRNAs, suggested that the transcription of the A. vinosum puf operon is initiated at 74 bp downstream from the bchZstop codon (295 bp upstream from the pufB (1) start codon), and regulated by a promoter located at its direct upstream. The possible promoter is overlapped with a binding motif of a repressor protein for pigment-biosynthesis genes, PpsR or CrtJ, known in other purple bacteria. No other possible promoters were found within the puf genes. These findings indicate that three sets of pufA and pufB genes of A. vinosum are co-transcribed as a long mRNA containing all the puf genes, and, from this long mRNA, the five short mRNAs are possibly derived by post-transcriptional modifications.

Journal Article↗
PubMed1974

Mössbauer spectroscopy of the nitrogenase proteins from Klebsiella pneumoniae. Structural assignments and mechanistic conclusions.

The Mo-Fe protein and the Fe protein which together constitute the nitrogenase of Klebsiella pneumoniae were prepared from bacteria grown in (57)Fe-enriched medium. The Mössbauer spectrum of the Mo-Fe protein, as isolated in the presence of Na(2)S(2)O(4), showed that the protein contained three iron species, called M4, M5 and M6. The area of the spectrum associated with species M4, with delta=0.65mm/s and DeltaE=3.05mm/s at 4.2 degrees K, corresponded to two iron atoms/molecule of protein and it is interpreted as being due to a high-spin ferrous, spin-coupled pair of iron atoms. The iron atoms of species M4 may be involved in the quaternary structure of the protein. Species M5, with delta=0.61mm/s and DeltaE=0.83mm/s at 77 degrees K, corresponded to eight iron atoms/molecule of protein and is interpreted as being due to Fe(4)S(4) or Fe(2)S(2) low-spin ferrous iron clusters. Species M6, with delta=0.37mm/s and DeltaE=0.71mm/s at 77 degrees K, also corresponded to eight iron atoms/molecule of protein and, at 4.2 degrees K, became a broad shallow absorption, characteristic of magnetic hyperfine interaction. Oxidation of the Mo-Fe protein with the redox dye Lauth's Violet did not affect the activity of the protein but changed species M4, M5 and M6 into the species M1 (delta=0.37mm/s, DeltaE=0.75mm/s at 77 degrees K, broad magnetic component at 4.2 degrees K) and M2 (delta=0.35mm/s, DeltaE=0.9mm/s at 4.2 degrees K). In the presence of the Fe protein, Na(2)S(2)O(4), ATP and Mg(2+), the M6 component of the Mo-Fe protein was replaced by species M7 with delta=0.46mm/s, DeltaE=1.04mm/s at 4.2 degrees K. The change in Mössbauer parameters associated with the M6 --> M7 transformation was very similar to the change observed on reduction of the high-potential Fe protein from Chromatium vinosum. In contrast, Na(2)S(2)O(4)-reduced Fe protein contained only one type of iron cluster (F4). Species F4 had delta=0.50mm/s, DeltaE=0.9mm/s at 195 degrees K, and at 4.2 degrees K broadened in a manner characteristic of a magnetic hyperfine interaction, associated with half-integral spin, equally distributed over all four atoms of the Fe protein. The Mössbauer spectra of the Mo-Fe and the Fe protein under argon were unaffected by the reducible substrates N(2) and C(2)H(2) and the inhibitor CO in the presence of ATP, Mg(2+) and Na(2)S(2)O(4). A number of Mössbauer spectral species associated with inactivated Mo-Fe and Fe proteins are described and discussed.

Adenosine Triphosphate↗
PubMed1972

Structure and properties of a synthetic analogue of bacterial iron--sulfur proteins.

The compound (Et(4)N)(2)[Fe(4)S(4)(SCH(2)Ph)(4)] has been prepared and its structure determined by x-ray diffraction. The Fe(4)S(4) core of the anion possesses a configuration of D(2d) symmetry that is closely related to the Fe(4)S(4) active-site structures of the high-potential iron protein from Chromatium and the ferredoxin from Micrococcus aerogenes. Electronic properties of the tetrameric anion have been partially characterized by measurement of proton magnetic resonance, Mössbauer, photoelectron, and electronic spectra, and magnetic susceptibility. Comparison of corresponding properties of [Fe(4)S(4)(SCH(2)Ph)(4)](2-) and the proteins implies that the oxidation levels of the synthetic tetramer, the reduced form of the high-potential protein, and the oxidized form of the 8-Fe ferredoxins are equivalent. The tetramer possesses the one-electron redox capacity associated with the 4-Fe centers of the ferredoxins. The structural and collective electronic features of [Fe(4)S(4)(SCH(2)Ph)(4)](2-) reveal it to be the first well-defined synthetic analogue of the active site of an iron-sulfur protein.

Bacterial Proteins↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed1993

Lead-catalyzed cleavage of ribonuclease P RNA as a probe for integrity of tertiary structure.

Pb(2+)-catalyzed cleavage of RNA has been shown previously to be a useful probe for tertiary structure. In the present study, Pb2+ cleavage patterns were identified for ribonuclease P RNAs from three phylogenetically disparate organisms, Escherichia coli, Chromatium vinosum, Bacillus subtilis, and for E. coli RNase P RNAs that had been altered by deletions. Each of the native RNAs undergoes cleavage at several sites in the core structure that is common to all bacterial RNase P RNAs. All the cleavages occur in non-paired regions of the secondary structure models of the RNAs, in regions likely to be involved in tertiary interactions. Two cleavage sites occur at homologous positions in all the native RNAs, regardless of sequence variation, suggesting common tertiary structural features. The Pb2+ cleavage sites in four deletion mutants of E. coli RNase P RNA differed from the native pattern, indicating alterations in the tertiary structures of the mutant RNAs. This conclusion is consistent with previously characterized properties of the mutant RNAs. The Pb2+ cleavage assay is thus a useful probe to reveal alteration of tertiary structure in RNase P RNA.

Base Sequence↗
PubMed1978

Amino acid sequence of a ferredoxin from Chlorobium thiosulfatophilum strain Tassajara, a photosynthetic green sulfur bacterium.

We have determined the amino acid sequence of a ferredoxin from a photosynthetic green sulfur bacterium, Chlorobium thiosulfatophilum strain Tassajara. It contains 61 amino acid residues with 9 cysteines, and 8 of the 9 were located at positions corresponding to those in clostridial-type ferredoxins. Other structural features were closer to those of ferredoxins from another photosynthetic bacterium, C. limicola, than to those of non-photosynthetic bacteria. Compared with ferredoxin from Chromatium, a photosynthetic purple sulfur bacterium, all photosynthetic bacterial ferredoxins have a common region in the carboxyl-terminal half with several extra residues and a unique cysteine residue. We compared all the photosynthetic bacterial ferredoxins that have been sequenced and concluded that C. thiosulfatophilum ferredoxin is most closely related to C. limicola ferredoxin I.

Amino Acid Sequence↗
PubMed1981

Siroheme as an active catalyst in sulfite reduction.

Siroheme extracted by acetone/HCl treatment of sulfite reductase from yeast and purified by column chromatography catalyzed the reduction of sulfite to thiosulfate and sulfide when coupled with a hydrogen-hydrogenase-methyl viologen system. The activity increased with decrease in pH from 7 to 4, and an apparent Km value of 50 mM for sulfite was obtained. In contrast to sirohydrochlorin plus Fe2+, addition of inorganic iron or 2,2'-bipyridine prior to the reduction reaction had scarcely any effect on the sulfite-reducing activity of siroheme. Hydroxylamine was reduced by siroheme at a much faster rate than sulfite, and the rate increased with increase in pH from 6 to 9. Siroheme extracted from Chromatium vinosum strain D sulfite reductase also reduced sulfite to thiosulfate and sulfide.

Catalysis↗
PubMed1985

Magnetic susceptibility of hydrogenase from Desulfovibrio vulgaris.

Magnetization and magnetic susceptibility measurements revealed that the hydrogenase [EC 1.12.2.1] from Desulfovibrio vulgaris Miyazaki F has an independent unpaired electron in its iron-sulfur cluster. The paramagnetic center of the Desulfovibrio hydrogenase is, therefore, different from that in the Chromatium hydrogenase which interacts with another paramagnetic center, probably nickel.

Desulfovibrio↗
PubMed1986

Interaction, functional relations and evolution of large and small subunits in Rubisco from prokaryota and eukaryota.

In early biological evolution anoxygenic photosynthetic bacteria may have been established through the acquisition of ribulose bisphosphate carboxylase-oxygenase (Rubisco). The establishment of cyanobacteria may have followed and led to the production of atmospheric oxygen. It has been postulated that a unicellular cyanobacterium evolved to cyanelles which were evolutionary precursors of chloroplasts of both green and non-green algae. The latter probably diverged from ancestors of green algae as evidenced by the occurrence of large (L) and small (S) subunit genes for Rubisco in the chloroplast genome of the chromophytic algae Olisthodiscus luteus. In contrast, the gene for the S subunit was integrated into the nucleus in the evolution of green algae and higher plants. The evolutionary advantages of this integration are uncertain because the function of S subunits is unknown. Recently, two forms of Rubisco (L8 and L8S8) of almost equivalent carboxylase and oxygenase activity have been isolated from the photosynthetic bacterium Chromatium vinosum. This observation perpetuates the enigma of S subunit function. Current breakthroughs are imminent, however, in our understanding of the function of catalytic L subunits because of the application of deoxyoligonucleotide-directed mutagenesis. Especially interesting mutated Rubisco molecules may have either enhanced carboxylase activity or higher carboxylase:oxygenase ratios. Tests of expression, however, must await the insertion of modified genes into the nucleus and chloroplasts. Methodology to accomplish chloroplast transformation is as yet unavailable. Recently, we have obtained the first transformation of cyanobacteria by a colE1 plasmid. We regard this transformation as an appropriate model for chloroplast transformation.

Bacteria↗
PubMed1993

Arhodomonas aquaeolei gen. nov., sp. nov., an aerobic, halophilic bacterium isolated from a subterranean brine.

Arhodomonas aquaeolei gen. nov., sp. nov., isolated from a petroleum reservoir production fluid, is described. The single isolate was an obligately halophilic, aerobic, gram-negative, oval rod-shaped bacterium that was actively motile by means of a single polar flagellum. It was catalase and oxidase positive. The isolate had a specific requirement for NaCl; growth occurred at NaCl concentrations between 6 and 20%, and optimal growth occurred in the presence of 15% NaCl. This species metabolized primarily organic acids and required biotin for growth. The name Arhodomonas is proposed for the new genus, which was placed in the gamma subclass of the Proteobacteria on the basis of the results of a 16S rRNA sequence analysis. Although A. aquaeolei is most closely related to purple sulfur bacteria (the genera Ectothiorhodospira and Chromatium), it is not a phototrophic microorganism, which is consistent with its isolation from a subterranean environment. The major components of its cellular fatty acids were C16:0, C18:1, C19:0, C16:1, and C18:0 acids. The DNA base composition of the type strain is 67 mol% G+C. The type and only strain is strain HA-1 (= ATCC 49307).

Bacterial Typing Techniques↗
PubMed1995

Molecular genetic analysis of a thioredoxin gene from Thiobacillus ferrooxidans.

The Thiobacillus ferrooxidans thioredoxin gene, trxA, was isolated by its ability to complement an Escherichia coli gshA trxA mutant which was otherwise unable to grow on minimal medium lacking glutathione. The T. ferrooxidans thioredoxin also enabled the in vivo reduction by E. coli of methionine sulfoxide to methionine, as well as the in vitro reduction of insulin. When present in E. coli, the T. ferrooxidans thioredoxin supported the replication of phage T7, but not the growth of phage M13. The T. ferrooxidans trxA gene was sequenced and the thioredoxin was found to be most like that of E. coli (71% identity) and Chromatium vinosum (70% identity). As in the case of E. coli, the gene was located immediately upstream of the gene for the rho transcriptional terminator. DNA:RNA blot hybridization and primer-extension analysis of the trxA gene in T. ferrooxidans and the cloned gene in E. coli indicated that it was transcribed as an independent unit and that the major transcriptional start sites were the same in both organisms.

Bacteriophage M13↗
PubMed1968

Properties of ribulose-1,5-diphosphate carboxylase (carboxydismutase) from Chinese cabbage and photosynthetic microorganisms.

Ribulose-1,5-diphosphate carboxylase (carboxydismutase) was prepared from Chinese Cabbage [Brassica petsai (Parl)] and the K(m) values and molecular weight were determined. These parameters were found to be in good agreement with values reported for this enzyme from other higher plants. Investigation of carboxydismutase activity from the photosynthetic micro-organisms Chlamydomonas reinhardi (IU 89+), Plectonema boryanum (IU 594), and Chromatium strain D showed striking similarity to the higher plant enzyme, when the sedimentation coefficients were compared.

Carboxy-Lyases↗
PubMed1975

Isotope Fractionation in Photosynthetic Bacteria during Carbon Dioxide Assimilation.

The delta (PDB) (13)C values have been determined for the cellular constituents and metabolic intermediates of autotrophically grown Chromatium vinosum. The isotopic composition of the HCO(3) (-) in the medium and the carbon isotopic composition of the bacterial cells change with the growth of the culture. The delta (PDB) (13)C value of the HCO(3) (-) in the media changes from an initial value of -6.6 per thousand to +8.1 per thousand after 10 days of bacterial growth and the delta (PDB) (13)C value of the bacterial cells change from -37.5 per thousand to -29.2 per thousand in the same period. The amount of carbon isotope fractionation during the synthesis of hexoses by the photoassimilation of CO(2) has a range of -15.5 per thousand at time zero to -22.0 per thousand after 10 days. This range of fractionation compares to the range of carbon isotope fractionation for the synthesis of sugars from CO(2) by ribulose 1,5-diphosphate carboxylase and the Calvin cycle.The amount of carbon isotope fractionation during the synthesis of aspartic acid from CO(2) is -24.9 per thousand at time zero and -15.0 per thousand after 10 days of bacterial growth. This amount of fractionation is in the range of carbon isotope fractionation for the synthesis of C(4) amino acids by a double carboxylation through ribulose 1,5-diphosphate and phosphoenolpyruvate carboxylase.

Journal Article↗
PubMed1976

Microencapsulation of chloroplast particles.

Chloroplast and photosystem I particles were encapsulated in small spheres (about 20 mum diameter) with an artificial membrane built up by cross-linking amino groups of protamine with toluenediisocyanate. The artificial membrane was permeable to small substrate and product molecules but not to soluble proteins. Photosystem I activity was retained by the encapsulated chloroplast particles. Washed photosystem I particles were encapsulated with the soluble proteins, ferredoxin, and ferredoxin-NADP oxidoreductase, and the microcapsules photoreduced NADP using ascorbate plus dichlorophenolindophenol as the electron donor. The photosystem I particles were also encapsulated with hydrogenase from Chromatium and a very low rate of photoevolution of hydrogen was obtained. The results show that chloroplast membrane fragments can be encapsulated with soluble proteins that couple transfer reactions to the primary photochemical apparatus.

Journal Article↗
PubMed1986

Effect of Betaine on Enzyme Activity and Subunit Interaction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Aphanothece halophytica.

The presence of betaine, a quaternary ammonium compound, at a concentration (0.5 molar) reported to accumulate inside Aphanothece halophytica in response to increasing external salinity, slightly promoted ribulose-1,5-bisphosphate (RuBP) carboxylase activity. KCl at 0.25 molar inhibited RuBP carboxylase about 55%. Betaine relieved the inhibition by 0.25 m KCl and the original uninhibited activity was restored at 1 m betaine. Other osmoregulatory solutes such as sucrose and glycerol also reduced KCl inhibition, though to a lesser extent than betaine. Proline had no effect. The protective effect of betaine against KCl inhibition of RuBP carboxylase activity was also observed in other cyanobacteria, i.e. Synechococcus ACMM 323, Plectonema boryanum, and Anabaena variabilis, and in the photosynthetic bacterium Rhodospirillum rubrum but not in Chromatium vinosum. Apart from betaine, other quaternary ammonium compounds, i.e. sarcosine and trimethylamine-N-oxide (TMAO), but not glycine, also protected the enzyme against KCl inhibition and the effectiveness of such compounds appeared to correlate with the extent of N-methylation. Heat and cold inactivation of the enzyme could be protected by either betaine or KCl. However, best protection occurred when both betaine and KCl were present together. The K(m) (CO(2)) was not altered by either betaine or KCl, nor when they were present together. However, the K(m) (RuBP) was increased about 5-fold by KCl, but was unaffected by betaine. The presence of betaine together with KCl lowered the KCl-raised K(m) (RuBP) by about half. The extent of the dissociation of the enzyme molecule under the condition of low ionic strength was reduced by either betaine or KCl alone and more so when they were present together. Glycine, sarcosine, and TMAO were more effective than betaine or KCl in lowering the extent of the dissociation of the enzyme molecule.

Journal Article↗