the phylogenetic position of hydrogenobacter acidophilus based on 16s rrna sequence analysis.hydrogenobacter acidophilus strain 3h-1 is a thermoacidophilic, obligately chemolithoautotrophic, hydrogen-oxidizer isolated from a japanese solfataric field. strain 3h-1 requires elemental sulfur for growth. we used pcr to amplify the 16s rrna gene of strain 3h-1, and sequenced the amplification product directly. phylogenetic analyses show strain 3h-1 is closely related to aquifex pyrophilus and may be located in the deepest branch within the eubacterial phylogenetic tree. sulfur-dependency of ...19948039650
amino acid sequence of cytochrome c-552 from a thermophilic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus.the complete amino acid sequence of cytochrome c-552 from an extremely thermophilic hydrogen bacterium, hydrogenobacter thermophilus tk-6 (iam 12695), was determined. it is a single polypeptide chain of 80 residues, and its molecular weight, including heme, was calculated to be 7,599. the sequence of cytochrome c-552 from h. thermophilus tk-6 closely resembles that of cytochromes c-551 from pseudomonas species. moreover, the tertiary structure of hydrogenobacter cytochrome c-552 is suggested to ...19892536668
thermostability of cytochrome c-552 from the thermophilic hydrogen-oxidizing bacterium hydrogenobacter thermophilus.the denaturation of the c-type cytochrome of the thermophilic bacterium hydrogenobacter thermophilus cytochrome c-552 by heat and guanidine hydrochloride was studied by measuring the change in circular dichroic spectra. the melting temperature (t1/2) of cytochrome c-552 in the presence of 1.5 m guanidine hydrochloride was 34 degrees c higher than that of the c-type cytochrome of pseudomonas aeruginosa cytochrome c-551. hydrogenobacter cytochrome c-552 is a much more stable protein than cytochrom ...19892558725
2-methylthio-1,4-naphthoquinone, a unique sulfur-containing quinone from a thermophilic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus.a quinone was extracted and purified from the cells of an extremely thermophilic hydrogen bacterium, hydrogenobacter thermophilus tk-6 (iam 12695). its chemical structure was determined as 2-methylthio-3-vi, vii-tetrahydromultiprenyl-1,4-naphthoquinone by elemental analysis, 1h nuclear magnetic resonance spectroscopy, mass spectroscopy, and infrared spectroscopy of the quinone and by gas-liquid chromatography and gas chromatography-mass spectroscopy analysis of the ozonolysis products of the qui ...19873584059
crystallization and preliminary x-ray diffraction study of cytochrome c552 from hydrogenobacter thermophilus.cytochrome c552 from a thermophilic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus, exhibits remarkable thermostability. the oxidized cytochrome c552 has been crystallized in an ethanol/water mixture by means of the vapor diffusion method. the crystals belong to the orthorhombic system, space group p2(1)2(1)2, with unit cell dimensions of a = 93.4 a, b = 52.9 a, and c = 32.4 a. most probably the asymmetric unit contains two molecules of cytochrome c552. the crystals diffract x-rays t ...19911665487
novel linear and branched polyamines in the extremely thermophilic eubacteria thermoleophilum, bacillus and hydrogenobacter.novel tertiary branched tetra-amines, quaternary branched penta-amines, linear penta-amines and linear hexa-amines were distributed as the major polyamines in six obligately extremely thermophilic eubacteria belonging to thermoleophilum, bacillus or hydrogenobacter. the major polyamine of thermoleophilum album and thermoleophilum minutum was identified as a quaternary branched penta-amine, 4,4-bis(3-aminopropyl)-1,8-diamino-4-azaoctane (nh2[ch2]3n+([ch2]3nh2)2[ch2]4nh2) by h.p.l.c., t.l.c. and g ...19921622392
molecular and functional properties of ef-tu from calderobacterium hydrogenophilum.protein synthesis elongation factor tu (ef-tu) was purified from an extreme thermophilic hydrogen-oxidizing bacterium calderobacterium hydrogenophilum. the relative molecular mass of ef-tu. gdp was 51,000. the factor was heat stable and lost only 50% of its activity after heating at 80 degrees c for 5 min. native and reduced ef-tu or ef-tu. gdp contained one sh-reactive group. the elongation factors from c. hydrogenophilum and e. coli were shown to be immunologically identical. from the southern ...19921414127
purification and characterization of atp:citrate lyase from hydrogenobacter thermophilus tk-6.atp:citrate lyase [atp citrate (pro-3s)-lyase; ec] was purified and characterized from the cells of hydrogenobacter thermophilus, an aerobic, thermophilic, hydrogen-oxidizing bacterium which fixes carbon dioxide by a reductive carboxylic acid cycle. the enzyme was quite stable, even in the absence of sulfhydryl reagents. optimum ph for reaction was 6.7 to 6.9, and optimum temperature was around 80 degrees c. the molecular weight of native enzyme was estimated to be 260,000 by gel filtrat ...19892703459
13c-nmr study of autotrophic co2 fixation in thermoproteus neutrophilus.the pathway of autotrophic co2 fixation has been investigated in the extremely thermophilic sulfur-respiring anaerobic archaebacterium thermoproteus neutrophilus. [1,4-13c2]succinate was used as a tracer since this compound was incorporated in small amounts virtually into all cell compounds without affecting the organism's ability to synthesize all cell constituents from co2. three representative amino acids, glutamate, aspartate and alanine were isolated from cells after growth for several gene ...19892506014
purification and characterization of pyruvate:ferredoxin oxidoreductase from hydrogenobacter thermophilus tk-6.pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, hydrogenobacter thermophilus tk-6, by precipitation with ammonium sulfate and fractionation by deae-sepharose cl-6b, polyacrylate-quaternary amine, hydroxyapatite, and superdex-200 chromatography. the native enzyme had a molecular mass of 135 kda and was composed of four different subunits with apparent molecular masses of 4 ...19979094223
dna-dependent rna polymerase from an extremely thermophilic hydrogen-oxidizing bacterium calderobacterium hydrogenophilum.extremely thermophilic bacterium calderobacterium hydrogenophilum contains dna-dependent rna polymerase with unusual properties. purified enzyme is thermoresistant (40 min at 100 degrees c) and exhibits similar subunit composition as eubacterial rna polymerases (e.g. escherichia coli). however, the enzyme is not susceptible to antibiotics which inhibit eubacterial rna polymerases (rifampicin and streptolydigin). the activity of the enzyme is inhibited by actinomycin d, daunomycin and heparin.19912025266
solution structure of thermostable cytochrome c-552 from hydrogenobacter thermophilus determined by 1h-nmr spectroscopy.the solution structure of a thermostable cytochrome c-552 from a thermophilic hydrogen oxidizing bacterium hydrogenobacter thermophilus was determined by proton nuclear magnetic resonance spectroscopy. twenty structures were calculated by the x-plor program on the basis of 902 interproton distances, 21 hydrogen bonds, and 13 torsion angle constraints. the pairwise average root-mean-square deviation for the main chain heavy atoms was 0.91 +/- 0.11 a. the main chain folding of the cytochrome c-552 ...19989657676
selected mutations in a mesophilic cytochrome c confer the stability of a thermophilic counterpart.mesophilic cytochrome c(551) of pseudomonas aeruginosa (pa c(551)) became as stable as its thermophilic counterpart, hydrogenobacter thermophilus cytochrome c(552) (ht c(552)), through only five amino acid substitutions. the five residues, distributed in three spatially separated regions, were selected and mutated with reference to the corresponding residues in ht c(552) through careful structure comparison. thermodynamic analysis indicated that the stability of the quintuple mutant of pa c(551) ...200010918067
conversion of a c type cytochrome to a b type that spontaneously forms in vitro from apo protein and heme: implications for c type cytochrome biogenesis and folding.cytochrome c(552) from hydrogenobacter thermophilus, a thermophilic bacterium, has been converted into a b type cytochrome, after mutagenesis of both heme-binding cysteines to alanine and expression in the cytoplasm of escherichia coli. the b type variant is less stable, with the guanidine hydrochloride unfolding midpoint occurring at a concentration 2 m lower than for the wild-type protein. the reduction potential is 75 mv lower than that of the recombinant wild-type protein. the heme can be re ...200010792037
elongation factor tu of the extreme thermophilic hydrogen oxidizing bacterium calderobacterium hydrogenophilum.protein synthesis elongation factor tu has been purified from an extreme thermophilic hydrogen oxidizing bacterium calderobacterium hydrogenophilum. the molecular mass of ef-tu. gdp is 51,000. the factor is heat stable and loses only 50% of its activity after heating for 5 min at 80 degrees c. under mild conditions trypsin cleaved ef-tu. gdp to four main fragments. only one fragment of mr = 20,000 had a mobility similar to the trypsin fragment "b" of escherichia coli ef-tu. other peptide fragmen ...19883415694
phylogenetic evidence for the existence of novel thermophilic bacteria in hot spring sulfur-turf microbial mats in sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in japan. however, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. this study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16s rrna genes amplified from the bulk dn ...19989572936
cloning, nucleotide sequence and expression of the cytochrome c-552 gene from hydrogenobacter thermophilus.a cytochrome c-552 gene from a thermophilic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus, was cloned by using two oligonucleotide probes, which had been synthesized based on the known amino acid sequence of the protein. a 780-bp psti-sphi fragment of the cloned dna was sequenced and found to contain the entire structural gene coding for cytochrome c-552 bracketed by apparent escherichia coli consensus sequences for initiation and termination of transcription. cytochrome c-552 is sy ...19911645652
stabilization of pseudomonas aeruginosa cytochrome c(551) by systematic amino acid substitutions based on the structure of thermophilic hydrogenobacter thermophilus cytochrome c(552).a heterologous overexpression system for mesophilic pseudomonas aeruginosa holocytochrome c(551) (pa c(551)) was established using escherichia coli as a host organism. amino acid residues were systematically substituted in three regions of pa c(551) with the corresponding residues from thermophilic hydrogenobacter thermophilus cytochrome c(552) (ht c(552)), which has similar main chain folding to pa c(551), but is more stable to heat. thermodynamic properties of pa c(551) with one of three singl ...199910608805
purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, hydrogenobacter thermophilus tk-6.2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, hydrogenobacter thermophilus tk-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by deae-sepharose cl-6b, polyacrylate-quaternary amine, hydroxyapatite, and superdex-200 chromatography. the purified enzyme had a molecular mass of about 105 kda and comprised two subunits (70 kda and 35 kda). the activity of the 2-oxoglutarate:ferredoxin oxid ...19968655524
purification and characterization of membrane-bound hydrogenase from hydrogenobacter thermophilus strain tk-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium.a membrane-bound hydrogenase was purified to electrophoretic homogeneity from the cells of hydrogenobacter thermophilus strain tk-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium. solubilization and purification were done aerobically in the presence of triton x-100. three chromatography steps were done for purification; butyl-sepharose, mono-q, and superose 6, in this order. purification was completed with 6.73% yield of total activity and with 21.4-fold increase of speci ...200010803945
cytochromes c-552 from two strains of the hydrogenotrophic bacterium alcaligenes eutrophus are sequence homologs of the cytochromes c8 from the denitrifying pseudomonads.soluble cytochromes c-552 were purified from two strains of the hydrogenothrophic species alcaligenes eutrophus and their amino acid sequences determined. the two cytochromes were found to have 5 differences out of a total of 89 residues. the proteins are clearly related to the cytochromes c8 (formerly called pseudomonas cytochromes c-551), but require a single residue insertion after the methionine sixth heme ligand relative to the pseudomonas aeruginosa protein. the consensus residues trp56 an ...19979434105
an escherichia coli ccm (cytochrome c maturation) deletion strain substantially expresses hydrogenobacter thermophilus cytochrome c552 in the cytoplasm: availability of haem influences cytochrome c552 maturation.the maturation of hydrogenobacter thermophilus cytochrome c552 in the cytoplasm of escherichia coli is unique among bacterial c-type cytochromes. it is now shown to be matured in a strain lacking the whole set of ccm (cytochrome c maturation) genes that are normally required for c-type cytochrome biogenesis in e. coli. as this cytochrome is thermostable we propose that the apocytochrome c552 has sufficient tertiary structure to allow the haem to slot into its binding pocket, which in turn trigge ...19989561726
loss of either of the two heme-binding cysteines from a class i c-type cytochrome has a surprisingly small effect on physicochemical properties.almost without exception, c-type cytochromes have heme covalently attached via two thioether linkages to the cysteine residues of a cxxch motif. the reasons for the covalent attachment are not understood. reported here is cytoplasmic expression in escherichia coli of axxch and cxxah variants of cytochrome c(552) from hydrogenobacter thermophilus; remarkably, the single thioether bond proteins have, apart from an altered visible absorption spectrum, almost identical properties, including thermal ...200010922364
a mutation blocking the formation of membrane or periplasmic endogenous and exogenous c-type cytochromes in escherichia coli permits the cytoplasmic formation of hydrogenobacter thermophilus holo cytochrome c552.a mutant of escherichia coli, jcb606, shown to be pleiotropically deficient in the formation of endogenous membrane and periplasmic c-type cytochromes, synthesised the apo form of the exogenous cytochrome c550 from paracoccus denitrificans, but not the holo form. in contrast, a cytoplasmically located holo form of hydrogenobacter thermophilus cytochrome c552 was found in e. coli jcb606. these findings support the proposition that the formation of the cytoplasmic h. thermophilus cytochrome c552 i ...19948187885
nitrate respiratory metabolism in an obligately autotrophic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus tk-6.hydrogenobacter thermophilus strain tk-6 was observed to grow anaerobically on nitrate as an electron acceptor when molecular hydrogen was used as an energy source. nitrite was detected as the product of a respiratory reaction. 15no, 15n2o, and 15n2 were detected with na15no3 as an electron acceptor. western immunoblot analysis showed that cell-free extracts from cells grown on nitrate reacted with antibodies against heme cd1-type nitrite reductase from pseudomonas aeruginosa. the positive bands ...200111271424
nmr and cd conformational studies of the c-terminal 16-peptides of pseudomonas aeruginosa c551 and hydrogenobacter thermophilus c552 cytochromes.the 16-amino acid sequences of the c-terminal helices of the homologous bacterial cytochromes c551 from pseudomonas aeruginosa and c552 from hydrogenobacter thermophilus were synthesized and their solution structure studied. circular dichroism and nmr experiments in aqueous solution have shown the presence of alpha-helices and 3(10)-helices. the populations of helical structures in phosphate buffer, ph 3.5, 293 k, were 21% for c551 and 20% for c552, but increased to 56.7 and 48%, respectively, i ...200111168887
novel division level bacterial diversity in a yellowstone hot spring.a culture-independent molecular phylogenetic survey was carried out for the bacterial community in obsidian pool (op), a yellowstone national park hot spring previously shown to contain remarkable archaeal diversity (s. m. barns, r. e. fundyga, m. w. jeffries, and n. r. page, proc. natl. acad. sci. usa 91:1609-1613, 1994). small-subunit rrna genes (rdna) were amplified directly from op sediment dna by pcr with universally conserved or bacteria-specific rdna primers and cloned. unique rdna types ...19989440526
hydrogenobacter subterraneus sp. nov., an extremely thermophilic, heterotrophic bacterium unable to grow on hydrogen gas, from deep subsurface geothermal water.a novel extreme thermophile was isolated from a water sample derived from a deep subsurface geothermal water pool at a depth of 1500 m in the hacchoubaru geothermal plant in oita prefecture, japan. the cells were found to be straight rods, each being motile by means of a polar flagellum. growth was observed at temperatures between 60 and 85 degrees c (optimum 78 degrees c; 120 min doubling time) and between ph 5.5 and ph 9.0 (optimum 7.5). the isolate was a strictly aerobic heterotroph capable o ...200111491342
phylogenetic position of the genus hydrogenobacter.the genus hydrogenobacter consists of extremely thermophilic, obligately chemolithotrophic organisms that exhibit anaerobic anabolism but aerobic catabolism. preliminary studies of the phylogenetic position of these organisms based on limited 16s ribosomal dna sequence data suggested that they belong to one of the earliest branching orders of the bacteria. in this study, the complete 16s ribosomal dna sequences of two type strains, hydrogenobacter thermophilus tk-6 and calderobacterium hydrogeno ...19947981093
molecular analysis of microbial community structure in an arsenite-oxidizing acidic thermal spring.electron microscopy (em), denaturing gradient gel electrophoresis (dgge) and 16s rdna sequencing were used to examine the structure and diversity of microbial mats present in an acid-sulphate-chloride (ph 3.1) thermal (58-62 degrees c) spring in norris basin, yellowstone national park, wy, usa, exhibiting rapid rates of arsenite oxidation. initial visual assessments, scanning em and geochemical measurements revealed the presence of three distinct mat types. analysis of 16s rdna fragments with dg ...200111578314
the cytochrome c fold can be attained from a compact apo state by occupancy of a nascent heme binding site.nmr techniques and 8-anilino-1-napthalenesulphonate (ans) binding studies have been used to characterize the apo state of a variant of cytochrome c(552) from hydrogenobacter thermophilus. in this variant the two cysteines that form covalent thioether linkages to the heme group have been replaced by alanine residues (c11a/c14a). cd studies show that the apo state contains approximately 14% helical secondary structure, and measurements of hydrodynamic radii using pulse field gradient nmr methods s ...200111584011
a novel five-subunit-type 2-oxoglutalate:ferredoxin oxidoreductases from hydrogenobacter thermophilus tk-6.a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus tk-6, fixes carbon dioxide via the reductive tca cycle. 2-oxoglutarate:ferredoxin oxidoreductase (ogor) of this strain is one of the key enzymes of the pathway. ogor of strain tk-6 has been reported to be a two-subunit-type ogor and encoded by korab. a gene cluster, fordabgef, encoding another ogor was found 148 bp upstream of korab in the opposite orientation. five of the for genes (fordabge) were r ...200211890705
the genes for anabolic 2-oxoglutarate: ferredoxin oxidoreductase from hydrogenobacter thermophilus tk-6.2-oxoglutarate: ferredoxin oxidoreductase (ogor) of a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus tk-6, is involved in carbon dioxide fixation via a reductive tca cycle. the enzyme is a heterodimer comprising subunits of 70 and 35 kda. the structural genes for the subunits (korab) were cloned with primers designed from n-terminal sequences of the purified enzyme. the korab genes were followed by two open reading frames of unknown function (orf3 ...200111401501
the escherichia coli cytochrome c maturation (ccm) system does not detectably attach heme to single cysteine variants of an apocytochrome c.cytochromes c are typically characterized by the covalent attachment of heme to polypeptide through two thioether bonds with the cysteine residues of a cys-xaa-xaa-cys-his peptide motif. in many gram-negative bacteria, the heme is attached to the polypeptide by the periplasmically functioning cytochrome c maturation (ccm) proteins. exceptionally, hydrogenobacter thermophilus cytochrome c(552), which has a normal cxxch heme-binding motif, and variants with axxch, cxxah, and axxah motifs, can be e ...200212048216
new isolates and physiological properties of the aquificales and description of thermocrinis albus sp. nov.the ecology of the aquificales was studied using a combination of phylogenetic and cultivation approaches. enrichment cultures were prepared from low-salt and marine samples of geothermally and volcanically heated environments of the united states (yellowstone national park), russia (kamchatka), italy, germany, djibouti, iceland, and africa (lake tanganyika). isolation of single cells using the selected cell cultivation technique resulted in 15 different pure cultures. comparisons of their 16s r ...200212215816
influence of amino acid side chain packing on fe-methionine coordination in thermostable cytochrome c.paramagnetic nmr and optical studies of the oxidized forms of mesophile pseudomonas aeruginosa cytochrome c(551) and its quintuple mutant (f7a/v13m/f34y/e43y/v78i), and thermophile hydrogenobacter thermophilus cytochrome c(552) demonstrated that the amino acid side chain packings in the protein interior influence the coordination bond between the heme iron and the axial methionine in the proteins. the strength of heme axial coordinations was found to correlate with the overall protein thermostab ...200212296704
characterization of hydrogenobacter thermophilus cytochromes c(552 )expressed in the cytoplasm and periplasm of escherichia coli.hydrogenobacter thermophilus cytochrome c(552) ( ht cyt c(552)) is a small monoheme protein in the cytochrome c(551) family. ht cyt c(552) is unique because it is hypothesized to undergo spontaneous cytoplasmic maturation (covalent heme attachment) when expressed in escherichia coli. this is in contrast to the usual maturation route for bacterial cytochromes c that occurs in the cellular periplasm, where maturation factors direct heme attachment. here, the expression of ht cyts c(552) in the per ...200211935350
microbial silica deposition in geothermal hot waters.a combined use of molecular ecological techniques and geochemical surveys revealed that thermophilic or hyperthermophilic microorganisms living in geothermal environments are likely to be implicated in the formation of biogenic siliceous deposits. electron microscopic observations indicated that numerous microorganism-like fabrics were preserved in naturally occurring siliceous deposits such as siliceous sinter, geyserite, and silica scale, which suggests microbial contribution to silica precipi ...200312664138
thermodynamic characterization of variants of mesophilic cytochrome c and its thermophilic counterpart.thermal stability was measured for variants of cytochrome c-551 (pa c-551) from a mesophile, pseudomonas aeruginosa, and a thermophilic counterpart, hydrogenobacter thermophilus cytochrome c-552 (ht c-552), by differential scanning calorimetry (dsc) at ph 3.6. the mutated residues in pa c-551, selected with reference to the corresponding residues in ht c-552, were located in three spatially separated regions: region i, phe7 to ala/val13 to met; region ii, glu34 to tyr/phe43 to tyr; and region ii ...200212082163
sulfurihydrogenibium subterraneum gen. nov., sp. nov., from a subsurface hot aquifer.a polyphasic taxonomic study was performed on a novel facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotroph recently isolated from subsurface hot aquifer water in a japanese gold mine. the cells were straight to slightly curved rods, with a single polar flagellum. growth was observed at 40-70 degrees c (optimum 60-65 degrees c; 80 min doubling time) and at ph 6.4-8.8 (optimum ph 7.5). the isolate was unable to use complex organic compounds, carbohyd ...200312807207
cytochrome c from a thermophilic bacterium has provided insights into the mechanisms of protein maturation, folding, and stability.cytochrome c is widely distributed in bacterial species, from mesophiles to thermophiles, and is one of the best-characterized redox proteins in terms of biogenesis, folding, structure, function, and evolution. experimental molecular biology techniques (gene cloning and expression) have become applicable to cytochrome c, enabling its engineering and manipulation. heterologous expression systems for cytochromes c in bacteria, for use in mutagenesis studies, have been established by extensive inve ...200212135473
cytochrome c maturation. the in vitro reactions of horse heart apocytochrome c and paracoccus dentrificans apocytochrome c550 with heme.c-type cytochromes are characterized by having the heme moiety covalently attached via thioether bonds between the heme vinyl groups and the thiols of conserved cysteine residues of the polypeptide chain. previously, we have shown the in vitro formation of hydrogenobacter thermophilus cytochrome c(552) (daltrop, o., allen, j. w. a., willis, a. c., and ferguson, s. j. (2002) proc. natl. acad. sci. u. s. a. 99, 7872-7876). in this work we report that thioether bonds can form spontaneously in vitro ...200312458205
stereoselective in vitro formation of c-type cytochrome variants from hydrogenobacter thermophilus containing only a single thioether vitro formation of hydrogenobacter thermophilus cytochrome c552 has previously been demonstrated (daltrop, o., allen, j. w. a., willis, a. c., and ferguson, s. j. (2002) proc. natl. acad. sci. u. s. a. 99, 7872-7876). now we report that the single cysteine variants of h. thermophilus c552, which bind heme via a single thioether bond, also form in vitro. furthermore, reaction of the apocytochromes containing either axxch or cxxah in the binding motif with 2-vinyldeuteroheme and 4-vinyldeuteroh ...200312707264
relationship between redox function and protein stability of cytochromes c.electrochemical, 1h nmr, and optical studies on mesophile pseudomonas aeruginosa cytochrome c551, its single (f34y) and quintuple (f7a/v13m/f34y/e43y/v78i) mutants, and thermophile hydrogenobacter thermophilus cytochrome c552 at wide temperature range demonstrated that the stable protein exhibits the low redox potential predominantly due to the enthalpic contribution to the redox reaction. the overall stability of the oxidized form was shown to determine the stability of the fe-methionine coordi ...200314599189
characterization of two different 2-oxoglutarate:ferredoxin oxidoreductases from hydrogenobacter thermophilus tk-6.a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus tk-6, fixes carbon dioxide via the reductive tca cycle. 2-oxoglutarate:ferredoxin oxidoreductase (ogor) is one of the key enzymes of this cycle. strain tk-6 has two distinct ogor enzymes termed for and kor. these enzymes were purified and characterized following heterologous expression in escherichia coli. the specific activity of for was approximately one-tenth of that of kor. additionally, for show ...200314652015
variation of the axial haem ligands and haem-binding motif as a probe of the escherichia coli c-type cytochrome maturation (ccm) system.cytochromes c are typically characterized by the covalent attachment of haem to polypeptide through two thioether bonds with the cysteine residues of a cys-xaa-xaa-cys-his peptide motif. in many gram-negative bacteria, the haem is attached to the polypeptide by the periplasmically functioning cytochrome c maturation (ccm) proteins. exceptionally, hydrogenobacter thermophilus cytochrome c552 can be expressed as a stable holocytochrome both in the cytoplasm of escherichia coli in an apparently unc ...200312901720
a novel enzyme, citryl-coa synthetase, catalysing the first step of the citrate cleavage reaction in hydrogenobacter thermophilus tk-6.we attempted to purify atp citrate lyase (acl) from hydrogenobacter thermophilus by following the citrate-, atp- and coa-dependent formation of an acyl-coa species that was detected as hydroxamate. however, citryl-coa rather than acetyl-coa was found, indicating that the purified enzyme was a novel citryl-coa synthetase (ccs) rather than acl. because the reaction catalysed by ccs corresponds to the first half of that mediated by acl, ccs may be responsible for citrate cleavage in h. thermophilus ...200415101981
a novel enzyme, citryl-coa lyase, catalysing the second step of the citrate cleavage reaction in hydrogenobacter thermophilus tk-6.a novel enzyme catalysing citryl-coa cleavage to acetyl-coa and oxaloacetate was purified from hydrogenobacter thermophilus tk-6, and designated citryl-coa lyase (ccl). the citrate cleavage reaction in this organism proceeded by a unique set of two consecutive reactions: (i). citryl-coa formation by citryl-coa synthetase (ccs) and (ii). citryl-coa cleavage by ccl. purified ccl gave a single 30 kda band in sds-page and gel filtration chromatography indicated that the native state of the enzyme ex ...200415101982
nmr analysis shows that a b-type variant of hydrogenobacter thermophilus cytochrome c552 retains its native structure.conversion of hydrogenobacter thermophilus cytochrome c(552) into a b-type cytochrome by mutagenesis of both heme-binding cysteines to alanines significantly reduces the stability of the protein (tomlinson, e. j., and ferguson, s. j. (2000) proc. natl. acad. sci. u. s. a. 97, 5156-5160). to understand the effects of this change on the structure and dynamics of the protein, hetero-nuclear (15)n-edited nmr techniques have been used to characterize this b-type variant. the backbone (15)n, (1)h(n), ...200414726539
chemical structure of a novel aminophospholipid from hydrogenobacter thermophilus strain tk-6.the phospholipid composition of hydrogenobacter thermophilus strain tk-6, an obligately chemolithoautotrophic, extremely thermophilic hydrogen bacterium, was analyzed. two of four phospholipids detected from the strain were assumed to be phosphatidylinositol and phosphatidylglycerol. an aminophospholipid named px, whose content among the phospholipids was 65%, was found to have a novel chemical structure by analysis of the dilyso form with nuclear magnetic resonance and fast atom bombardment-mas ...200111591674
a novel biotin protein required for reductive carboxylation of 2-oxoglutarate by isocitrate dehydrogenase in hydrogenobacter thermophilus tk-6.isocitrate dehydrogenase was purified from hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. the enzyme had similar structural properties to the isocitrate dehydrogenase of escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, ph dependency and kinetic parameters. notably, the enzyme catalysed the oxidative decarboxylation of isocitrate, but not the reductive carboxylation of 2-oxoglutarate. the carboxylation reaction required ...200414731279
purification and characterization of ferredoxin from hydrogenobacter thermophilus strain tk-6.ferredoxin was purified from cells of hydrogenobacter thermophilus strain tk-6. purification was performed aerobically by the addition of octyl-beta-glucoside to the buffers. the purified ferredoxin had a molecular mass of 13,000 and contained a [4f3-4s] cluster. the protein had a long stretch at the n-terminal region; however, the sequence was not similar to the sequences of ferredoxins with a long stretch from archaebacteria.19968987606
complete thermal-unfolding profiles of oxidized and reduced cytochromes c.the complete thermal-unfolding profiles of both oxidized and reduced forms of cytochrome c551 (pa) from mesophilic pseudomonas aeruginosa and cytochrome c552 (ht) from thermophilic hydrogenobacter thermophilus were obtained by the newly developed pressure-proof cell compartment installed in a circular dichroic spectrometer, which facilitates protein thermal-unfolding experiments up to 180 degrees c. the thermodynamic cycle, which relates protein stability and redox function, indicated that the r ...200415535669
hydrogenothermus marinus gen. nov., sp. nov., a novel thermophilic hydrogen-oxidizing bacterium, recognition of calderobacterium hydrogenophilum as a member of the genus hydrogenobacter and proposal of the reclassification of hydrogenobacter acidophilus as hydrogenobaculum acidophilum gen. nov., comb. nov., in the phylum 'hydrogenobacter/aquifex'.a novel thermophilic, hydrogen-oxidizing bacterium, vm1t, has been isolated from a marine hydrothermal area of vulcano island, italy. cells of the strain were gram-negative rods, 2-4 microm long and 1-1.5 microm wide with four to seven monopolarly inserted flagella. cells grew chemolithoautotrophically under an atmosphere of h2/co2 (80:20) in the presence of low concentrations of o2 (optimum 1-2%). carbohydrates and peptide substrates were not utilized, neither for energy generation nor as a sou ...200111594618
polyamine analysis for chemotaxonomy of thermophilic eubacteria: polyamine distribution profiles within the orders aquificales, thermotogales, thermodesulfobacteriales, thermales, thermoanaerobacteriales, clostridiales and bacillales.cellular polyamines of 45 thermophilic and 8 related mesophilic eubacteria were investigated by hplc and gc analyses for the thermophilic and chemotaxonomic significance of polyamine distribution profiles. spermidine and a quaternary branched penta-amine, n4-bis(aminopropyl)norspermidine, were the major polyamine in thermocrinis, hydrogenobacter, hydrogenobaculum, aquifex, persephonella, sulfurihydrogenibium, hydrogenothermus, balnearium and thermovibrio, located in the order aquificales. thermo ...200415747232
signature lipids and stable carbon isotope analyses of octopus spring hyperthermophilic communities compared with those of aquificales representatives.the molecular and isotopic compositions of lipid biomarkers of cultured aquificales genera have been used to study the community and trophic structure of the hyperthermophilic pink streamers and vent biofilm from octopus spring. thermocrinis ruber, thermocrinis sp. strain hi 11/12, hydrogenobacter thermophilus tk-6, aquifex pyrophilus, and aquifex aeolicus all contained glycerol-ether phospholipids as well as acyl glycerides. the n-c(20:1) and cy-c(21) fatty acids dominated all of the aquificale ...200111679343
plasticity of the protein folding landscape: switching between on- and off-pathway intermediates.proteins may fold via parallel routes partitioned by the relative effect of solvent conditions on the relevant transition states. thus, intermediates may or may not necessarily be obligatory species accumulated during the folding process, but rather kinetic traps due to the ruggedness of the folding landscape. implicit in this view is the notion of plasticity of the folding pathway: proteins can be rerouted through the energy landscape by mutational, topological or solvent perturbations. our wor ...200717658452
a new ecological adaptation to high sulfide by a hydrogenobacter sp. growing on sulfur compounds but not on hydrogen.thermophilic bacteria were isolated from a sulfide-rich, neutral hot spring in iceland on gelrite minimal medium with 16 mm thiosulfate. the isolates were aerobic, obligate chemolithoautotrophs and used thiosulfate and sulfur as electron donors, producing sulfate from both substrates. no growth was observed with hydrogen as the sole electron donor, and no hydrogenase activity was detected. the cells were gram-negative and usually single, 4-5 microm long and 0.7 microm in diameter and formed sulf ...200111372652
molecular signatures in protein sequences that are characteristics of the phylum aquificae.species of the phylum aquificae are of great interest due to their strict extreme thermophilic growth characteristics. presently, there is no known molecular characteristic which is unique to this group of bacteria. this work describes six conserved inserts and deletions (indels or signature sequences) in four widely distributed proteins that are distinctive features of species from the phylum aquificae. these include three signatures consisting of a 2 aa insert, a 5-6 aa insert and a 6 aa delet ...200616403873
two tandemly arranged ferredoxin genes in the hydrogenobacter thermophilus genome: comparative characterization of the recombinant [4fe-4s] ferredoxins.a thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus tk-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. a gene encoding a ferredoxin involved in this cycle as an electron donor (htfd1) was cloned and sequenced. interestingly, another ferredoxin gene (encoding htfd2) was found in tandem with the htfd1 gene. these two ferredoxin genes overlapped by four bp, and transcriptional analysis revealed that they are co-transc ...200515973049
signature sequences in diverse proteins provide evidence for the late divergence of the order aquificales.the aquificales species are presently believed to be the earliest branching lineage within bacteria. however, the branching order of this group in different phylogenetic trees is highly variable and not resolved. in the present work, the phylogenetic placement of aquificales was examined by means of a cladistic approach based on the shared presence or absence of definite signature sequences (consisting of conserved inserts or deletions) in many highly conserved and important proteins, e.g. rna p ...200415179606
photo-cidnp nmr spectroscopy of a heme-containing protein.there are relatively few examples of the application of photo-cidnp nmr spectroscopy to chromophore-containing proteins. the most likely reason for this is that simultaneous absorption of light by the photosensitiser molecule and the protein chromophore reduces the effectiveness of the photochemical reaction that produces the observed nuclear polarisation. we present details of experiments performed on the air-oxidised form of a small cytochrome, from the thermophilic bacterium hydrogenobacter t ...200515886032
[preliminary study on bacterial diversity in a hot spring by using culture-independent approach].bacterial biodiversity of the hot spring dagunguo in tengchong rehai in yunnan province, china was preliminarily studied with culture-independent method. five complete 16s rdna sequences were obtained and phylogenetically analysed. four of these sequences were closely related to the genera: bacillus, hydrogenobacter and pseudomonas, and the other one was clustered with the family thermodesulfobacteriaceae between an unpublished genus geothermobacterium and thermodesulfobacteria. the v8 high vari ...200316281549
an obligatory intermediate in the folding pathway of cytochrome c552 from hydrogenobacter thermophilus.the folding mechanism of many proteins involves the population of partially organized structures en route to the native state. identification and characterization of these intermediates is particularly difficult, as they are often only transiently populated and may play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. following different spectroscopic probes, and employing state-of-the-art kinetic analysis, we present evidence that the folding ...200515883159
microbial community in a geothermal aquifer associated with the subsurface of the great artesian basin, investigate the biomass and phylogenetic diversity of the microbial community inhabiting the deep aquifer of the great artesian basin (gab), geothermal groundwater gushing out from the aquifer was sampled and analyzed. microbial cells in the groundwater were stained with acridine orange and directly counted by epifluorescence microscopy. microbial cells were present at a density of 10(8)-10(9) cells per liter of groundwater. archaeal and bacterial small-subunit rrna genes (rdnas) were amplifi ...200515980939
synthesis of holo paracoccus denitrificans cytochrome c550 requires targeting to the periplasm whereas that of holo hydrogenobacter thermophilus cytochrome c552 does not. implications for c-type cytochrome biogenesis.expression from a plasmid of the complete gene, including the codons for the n-terminal periplasmic targeting signal, for cytochrome c550 of paracoccus denitrificans led to the formation of the holo protein in the periplasms of both p. denitrificans and escherichia coli. expression of the gene from which the region coding for the targeting signal had been specifically deleted resulted in formation of apo-protein in the cytoplasms of both organisms. these findings are consistent with haem attachm ...19948119410
anabolic five subunit-type pyruvate:ferredoxin oxidoreductase from hydrogenobacter thermophilus tk-6.the thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus tk-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. a gene cluster, poredabg, encoding pyruvate:ferredoxin oxidoreductase (por), which plays a key role in this cycle, was cloned and sequenced. the nucleotide sequence and the gene organization were similar to those of the five subunit-type 2-oxoglutarate:ferredoxin oxidoreductase from this strain, although the ana ...200616343420
structure of cytochrome c552 from a moderate thermophilic bacterium, hydrogenophilus thermoluteolus: comparative study on the thermostability of cytochrome c.we have studied the structure-thermostability relationship using cytochromes c from mesophilic and thermophilic bacteria; pseudomonas aeruginosa (pac(551)) growing at 37 degrees c and hydrogenobacter thermophilus (htc(552)) at 72 degrees c and showed that only five residues primarily differentiate their stabilities. for a more comprehensive study, we found hydrogenophilus thermoluteolus (pseudomonas hydrogenothermophila) growing at 52 degrees c and showed the moderate stability of the cytochrome ...200616681384
five amino acid residues responsible for the high stability of hydrogenobacter thermophilus cytochrome c552: reciprocal mutation analysis.five amino acid residues responsible for extreme stability have been identified in cytochrome c(552) (ht c(552)) from a thermophilic bacterium, hydrogenobacter thermophilus. the five residues, which are spatially distributed in three regions of ht c(552), were replaced with the corresponding residues in the homologous but less stable cytochrome c(551) (pa c(551)) from pseudomonas aeruginosa. the quintuple ht c(552) variant (a7f/m13v/y34f/y43e/i78v) showed the same stability against guanidine hyd ...200415598654
further enhancement of the thermostability of hydrogenobacter thermophilus cytochrome c552.thermophile hydrogenobacter thermophilus cytochrome c(552) (ht) is a stable protein with denaturation temperatures (t(m)) of 109.8 and 129.7 degrees c for the oxidized and reduced forms, respectively [uchiyama, s., ohshima, a., nakamura, s., hasegawa, j., terui, n., takayama, s. j., yamamoto, y., sambongi, y., and kobayashi, y. (2004) j. am. chem. soc. 126, 14684-14685]. the removal of a single hydroxyl group from the hydrophobic core of ht, through the replacement of a tyr by phe, resulted in f ...200616953587
purification, characterization, and gene cloning of thermophilic cytochrome cd1 nitrite reductase from hydrogenobacter thermophilus tk-6.a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus tk-6 can grow autotrophically under anaerobic conditions by denitrification. one of the denitrification enzymes, cytochrome cd(1) nitrite reductase, was isolated and its gene was cloned from strain tk-6. the subunit molecular mass of the purified enzyme was 61.5 kda and the isoelectric point was determined to be 9.3. the optimum temperature and ph for the enzymatic reaction were 70-75 degrees c and 6 ...200616781467
redox properties of wild-type and heme-binding loop mutants of bacterial cytochromes c measured by direct electrochemistry.we have used protein film voltammetry (pfv) to determine the midpoint potentials of the pseudomonas aeruginosa, hydrogenobacter thermophilus, and nitrosomonas europaea wild-type monoheme cytochromes c (cyts c; pa, ht, and ne, respectively), as well as pa n64q, ht q64n, and ne v65delta mutants, as a function of ph, and buffer conditions. recent studies have suggested that the identity of the 64 position of the heme-binding loop (either asn or gln) strongly influences the conformation of the met l ...200516296855
[bacterial diversity of disused thermal vents in tibetan yangbajing region].to study the bacterial diversity in sediment and water from two disused thermal vents in yangbajing, tibet, china.200919445178
suppression of axial methionine fluxion in hydrogenobacter thermophilus gln64asn cytochrome c552.proteins in the cytochrome c (cyt c) family with his-met heme axial ligation display diverse heme electronic structures as revealed by the nmr spectra of their oxidized (paramagnetic) forms. these variations in electronic structure are thought to result primarily from differences in heme axial met orientation among cyt c species. the factors determining met orientation in cyts c, however, remain poorly understood. an additional layer of complexity was revealed with the recent finding that the ax ...200515794659
role of two 2-oxoglutarate:ferredoxin oxidoreductases in hydrogenobacter thermophilus under aerobic and anaerobic conditions.hydrogenobacter thermophilus tk-6 is a thermophilic, hydrogen-oxidizing bacterium that fixes carbon dioxide as a sole carbon source via the reductive tricarboxylic acid cycle. 2-oxoglutarate:ferredoxin oxidoreductase (ogor) is one of the key enzymes in the pathway. strain tk-6 has at least two isozymes of ogor, namely for and kor. these ogors showed different expression patterns under aerobic conditions than under anaerobic conditions. in this work, we developed a homologous recombination method ...200616978355
heme axial methionine fluxionality in hydrogenobacter thermophilus cytochrome c552.the heme group in paramagnetic (s = 1/2) ferricytochromes c typically displays a markedly asymmetric distribution of unpaired electron spin density among the heme pyrrole beta substituents. this asymmetry is determined by the orientations of the heme axial ligands, histidine and methionine. one exception to this is ferricytochrome c(552) from hydrogenobacter thermophilus, which has similar amounts of unpaired electron spin density at the beta substituents on all four heme pyrroles. here, determi ...200415161973
gene structure and expression profile of cytochrome bc nitric oxide reductase from hydrogenobacter thermophilus tk-6.the genes for a nitric oxide reductase-like cytochrome bc complex were cloned from a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus tk-6. the structural genes norc and norb, which encode cytochrome c and cytochrome b subunits of the complex respectively, are probably transcribed as a tricistronic operon with a following gene encoding a putative membrane protein. norc has, unusually, two hydrophobic transmembrane spans in its n-terminus. immunoblot ...200616861801
in vitro studies on thioether bond formation between hydrogenobacter thermophilus apocytochrome c(552) with metalloprotoporphyrin derivatives.previously, in vitro formation of thioether bonds between hydrogenobacter thermophilus apocytochrome c(552) and fe-protoporphyrin ix has been demonstrated. now we report studies on the reaction between the metalloderivatives zn-, co-, and mn-protoporphyrin ix and the cysteine thiols of h. thermophilus apocytochrome c(552). all of these metalloporphyrins were capable of forming a "b-type cytochrome" state in which the hydrophobic prosthetic group is bound non-covalently. zn(ii)-protoporphyrin ix ...200415326165
novel enzyme reactions related to the tricarboxylic acid cycle: phylogenetic/functional implications and biotechnological applications.the tricarboxylic acid (tca) cycle is an energy-producing pathway for aerobic organisms. however, it is widely accepted that the phylogenetic origin of the tca cycle is the reductive tca cycle, which is a non-calvin-type carbon-dioxide-fixing pathway. most of the enzymes responsible for the oxidative and reductive tca cycles are common to the two pathways, the difference being the direction in which the reactions operate. because the reductive tca cycle operates in an energetically unfavorable d ...200717333169
molecular dynamics simulations of hydrogenobacter thermophilus cytochrome c552: comparisons of the wild-type protein, a b-type variant, and the apo state.molecular dynamic simulations have been performed for wild-type hydrogenobacter thermophilus cytochrome c(552), a b-type variant of the protein, and the apo state with the heme prosthetic group removed. in the b-type variant, cys 10 and cys 13 were mutated to alanine residues, and so the heme group was no longer covalently bound to the protein. two 8-ns simulations have been performed for each system at 298 and 360 k. the simulations of the wild-type protein at 298 k show a very close agreement ...200616981201
characterization of n-terminal amino group-heme ligation emerging upon guanidine hydrochloric acid induced unfolding of hydrogenobacter thermophilus ferricytochrome c552.nonnative heme coordination structures emerging upon guanidine hydrochloric acid (gdnhcl) induced unfolding of hydrogenobacter thermophilus ferricytochrome c552 were characterized by means of paramagnetic nmr. the heme coordination structure possessing the n-terminal amino group of the peptide chain in place of axial met (his-nterm form) was determined in the presence of gdnhcl concentrations in excess of 1.5 m at neutral ph. the stability of the his-nterm form at ph 7.0 was found to be comparab ...200817899223
local conformational transition of hydrogenobacter thermophilus cytochrome c552 relevant to its redox order to elucidate the molecular mechanisms responsible for the apparent nonlinear behavior of the temperature dependence of the redox potential of hydrogenobacter thermophilus cytochrome c552 [takahashi, y., sasaki, h., takayama, s. j., mikami, s., kawano, s., mita, h., sambongi, y., and yamamoto, y. (2006) biochemistry 45, 11005-11011], its heme active site structure has been characterized using variable-temperature and -pressure nmr techniques. the study revealed a temperature-dependent co ...200717658890
heme axial methionine fluxion in pseudomonas aeruginosa asn64gln cytochrome c551.heme axial methionine ligands in ferricytochromes c552 from hydrogenobacter thermophilus (ht) and nitrosomonas europaea, both members of the cyt c8 family, display fluxional behavior. the ligand motion, proposed to be inversion at sulfur, results in an unusually small range of hyperfine shifts for heme substituents in these proteins. herein, heme axial met fluxion is induced in a structurally homologous cytochrome c551 from pseudomonas aeruginosa (pa) by substituting heme pocket residue asn64 wi ...200516271000
5'-end maturation of trna in aquifex aeolicus.5'-end maturation of trna primary transcripts is thought to be ubiquitously catalyzed by ribonuclease p (rnase p), a ribonucleoprotein enzyme in the vast majority of organisms and organelles. in the hyperthermophilic bacterium aquifex aeolicus, neither a gene for the rna nor the protein component of bacterial rnase p has been identified in its sequenced genome. here, we demonstrate the presence of an rnase p-like activity in cell lysates of a. aeolicus. detection of activity was sensitive to the ...200818208351
effects of axial methionine coordination on the in-plane asymmetry of the heme electronic structure of cytochrome c.the paramagnetic susceptibility ( chi) tensors of the oxidized forms of thermophile hydrogenobacter thermophilus cytochrome c(552) (ht cyt c(552)) and a quintuple mutant (f7a/v13 m/f34y/e43y/v78i; qm) of mesophile pseudomonas aeruginosa cytochrome c(551) (pa cyt c(551)) have been determined on the basis of the redox-dependent (1)h nmr shift changes of the main-chain nh and c(alpha)h proton resonances of non-coordinated amino acid residues and the nmr structures of the reduced forms of the corres ...200415235942
methionine ligand lability of type i cytochromes c: detection of ligand loss using protein film voltammetry.protein film voltammetry (pfv) is used to interrogate the behavior of a variety of bacterial and mitochondrial his/met-ligated cytochromes c. while analogous studies upon alkanethiol-modified gold electrodes reveal the anticipated fe(ii/iii) couple only, pfv using pyrolytic graphite edge (pge) electrodes demonstrates the presence of a lower-potential form of each of the cyts c studied, with a potential of approximately -100 mv (vs hydrogen). the generation of the novel, lower-potential state is ...200818454519
nondecarboxylating and decarboxylating isocitrate dehydrogenases: oxalosuccinate reductase as an ancestral form of isocitrate dehydrogenase.isocitrate dehydrogenase (icdh) from hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. in this study, the oxidation reaction catalyzed by h. thermophilus icdh was kinetically analyzed. as a result, a rapid equilibrium random-order mechanism was suggested. the affinities of both substrates (isocitrate and nad+) toward the enzyme were extremely low ...200818203822
heme attachment motif mobility tunes cytochrome c redox potential.hydrogen exchange (hx) rates and midpoint potentials (em) of variants of cytochrome c from pseudomonas aeruginosa (pa cyt c551) and hydrogenobacter thermophilus (ht cyt c552) have been characterized in an effort to develop an understanding of the impact of properties of the cys-x-x-cys-his pentapeptide c-heme attachment (cxxch) motif on heme redox potential. despite structural conservation of the cxxch motif, ht cyt c552 exhibits a low level of protection from hx for amide protons within this mo ...200717900177
a soluble nadh-dependent fumarate reductase in the reductive tricarboxylic acid cycle of hydrogenobacter thermophilus tk-6.fumarate reductase (frd) is an enzyme that reduces fumarate to succinate. in many organisms, it is bound to the membrane and uses electron donors such as quinol. in this study, an frd from a thermophilic chemolithoautotrophic bacterium, hydrogenobacter thermophilus tk-6, was purified and characterized. frd activity using nadh as an electron donor was not detected in the membrane fraction but was found in the soluble fraction. the purified enzyme was demonstrated to be a novel type of frd, consis ...200818757546
stabilization mechanism of cytochrome c552 from a moderately thermophilic bacterium, hydrogenophilus thermoluteolus.cytochrome c(552) (ph c(552)) from moderately thermophilic hydrogenophilus thermoluteolus exhibits stability intermediate between those of cytochrome c(552) (ht c(552)) from thermophilic hydrogenobacter thermophilus and cytochrome c(551) (pa c(551)) from mesophilic pseudomonas aeruginosa. to understand the mechanism of stabilization of ph c(552), we introduced mutations into ph c(552) at five sites, which, in ht c(552), are occupied by the amino acids responsible for stability higher than the le ...200818685221
linking microbial oxidation of arsenic with detection and phylogenetic analysis of arsenite oxidase genes in diverse geothermal environments.the identification and characterization of genes involved in the microbial oxidation of arsenite will contribute to our understanding of factors controlling as cycling in natural systems. towards this goal, we recently characterized the widespread occurrence of aerobic arsenite oxidase genes (aroa-like) from pure-culture bacterial isolates, soils, sediments and geothermal mats, but were unable to detect these genes in all geothermal systems where we have observed microbial arsenite oxidation. co ...200919196273
stability of the heme fe-n-terminal amino group coordination bond in denatured cytochrome the denatured states of hydrogenobacter thermophilus cytochrome c(552) (ht) and pseudomonas aeruginosa cytochrome c(551) (pa), and their mutants, the n-terminal amino group of the polypeptide chain is coordinated to heme fe in place of the axial met, the his-n(term) form being formed. the coordination of the n-terminal amino group to heme fe leads to loop formation by the n-terminal stretch preceding the first cys residue bound to the heme, and the n-terminal stretches of ht and pa are differ ...200919053349
a novel ferredoxin-dependent glutamate synthase from the hydrogen-oxidizing chemoautotrophic bacterium hydrogenobacter thermophilus tk-6.glutamate synthases are classified according to their specificities for electron donors. ferredoxin-dependent glutamate synthases had been found only in plants and cyanobacteria, whereas many bacteria have nadph-dependent glutamate synthases. in this study, hydrogenobacter thermophilus, a hydrogen-oxidizing chemoautotrophic bacterium, was shown to possess a ferredoxin-dependent glutamate synthase like those of phototrophs. this is the first observation, to our knowledge, of a ferredoxin-dependen ...200717237175
ferredoxin-nadp reductase from the thermophilic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus tk-6.the thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, hydrogenobacter thermophilus tk-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. small iron-sulfur proteins, ferredoxins, play a central role as low-potential electron donors for this cycle. the fpr gene of this bacterium, encoding a putative ferredoxin-nadp(+) reductase (fnr, ec, was expressed in escherichia coli, and the recombinant protein was purified to homogeneity. unexpected ...200919552713
native and unfolded cytochrome c--comparison of dynamics using 2d-ir vibrational echo spectroscopy.unfolded vs native co-coordinated horse heart cytochrome c (h-cyt c) and a heme axial methionine mutant cyt c552 from hydrogenobacter thermophilus ( ht-m61a) are studied by ir absorption spectroscopy and ultrafast 2d-ir vibrational echo spectroscopy of the co stretching mode. the unfolding is induced by guanidinium hydrochloride (guhcl). the co ir absorption spectra for both h-cyt c and ht-m61a shift to the red as the guhcl concentration is increased through the concentration region over which u ...200818646797
effects of cysteine introduction into three homologous cytochromes c.a cysteine residue was systematically introduced into three homologous cytochromes c from hydrogenobacter thermophilus, hydrogenophilus thermoluteolus, and pseudomonas aeruginosa at a conserved position. the h. thermoluteolus variant showed the most decreased thermal stability as compared with the wild type, which might have been due in part to crosslinked polymer formation. the effects of cysteine introduction differed even at the conserved position in these homologous proteins.200919420693
carboxylation reaction catalyzed by 2-oxoglutarate:ferredoxin oxidoreductases from hydrogenobacter thermophilus.hydrogenobacter thermophilus tk-6 is a thermophilic, chemolithoautotrophic, hydrogen-oxidizing bacterium that fixes carbon dioxide via the reductive tricarboxylic acid (rtca) cycle. 2-oxoglutarate:ferredoxin oxidoreductase (ogor) is the key enzyme in this cycle that fixes carbon dioxide. the genome of strain tk-6 encodes at least two distinct ogor enzymes, termed for and kor. we report here a method for measuring the carboxylation of succinyl-coa catalyzed by ogors. the method involves the in vi ...201019894084
cytochrome c552 mutants: structure and dynamics at the active site probed by multidimensional nmr and vibration echo spectroscopy.spectrally resolved infrared stimulated vibrational echo experiments are used to measure the vibrational dephasing of a co ligand bound to the heme cofactor in two mutated forms of the cytochrome c552 from hydrogenobacter thermophilus. the first mutant (ht-m61a) is characterized by a single mutation of met61 to an ala (ht-m61a), while the second variant is doubly modified to have gln64 replaced by an asn in addition to the m61a mutation (ht-m61a/q64n). multidimensional nmr experiments determined ...200616986870
biomass production and energy source of thermophiles in a japanese alkaline geothermal pool.microbial biomass production has been measured to investigate the contribution of planktonic bacteria to fluxations in dissolved organic matter in marine and freshwater environments, but little is known about biomass production of thermophiles inhabiting geothermal and hydrothermal regions. the biomass production of thermophiles inhabiting an 85 degrees c geothermal pool was measured by in situ cultivation using diffusion chambers. the thermophiles' growth rates ranged from 0.43 to 0.82 day(-1), ...201019878264
correlation between the stability and redox potential of three homologous cytochromes c from two thermophiles and one mesophile.the stability of the oxidized and reduced forms of three homologous cytochromes c from two thermophiles and one mesophile was systematically monitored by means of soret absorption measurements in the presence of various concentrations of a denaturant, guanidine thiocyanate, at ph 7.0 at 25 degrees c. thermophilic hydrogenobacter thermophilus cytochrome c(552) was the most stable in both redox states, followed by moderately thermophilic hydrogenophilus thermoluteolus cytochrome c(552), and then m ...200919202290
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