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R K Thauer

Publications and source records attributed to R K Thauer.

At least 145 records · Page 8Linked to original sources
PubMed1989

The magnetic properties of the nickel cofactor F430 in the enzyme methyl-coenzyme M reductase of Methanobacterium thermoautotrophicum.

Cofactor 430 of methyl-coenzyme M reductase from Methanobacterium thermoautotrophicum was studied in both the extracted form in aqueous solution and protein-bound by using low-temperature magnetic-circular-dichroism spectroscopy. In both forms the nickel was present as high-spin paramagnetic nickel(II), spin S = 1, subject to almost equal zero-field splitting (cofactor F430, D = +9.0 cm-1, E/D = 0; methyl-coenzyme M reductase, D = +8.5 cm-1, [E/D[ = 0.2). This suggests identical axial co-ordination by oxygen ligand(s) both in aqueous cofactor F430 and in the investigated state of the protein.

Circular Dichroism↗
PubMed1989

Proton translocation coupled to the oxidation of carbon monoxide to CO2 and H2 in Methanosarcina barkeri.

Cell suspensions of acetate-grown Methanosarcina barkeri mediate the conversion of CO and H2O to CO2 and H2. The reaction is coupled with the phosphorylation of ADP. Evidence is presented that CO oxidation by the cells is associated with the transient acidification of the suspension medium. Up to 2 mol vectorial protons were measured/mol CO oxidized when the transmembrane electrical gradient was kept low by the addition of valinomycin (20 nmol/mg protein) and KCl (200 mM) or of KSCN (50 mM). No transient acidification was observed in the presence of the protonophore tetrachlorosalicylanilide which stimulated rather than inhibited CO oxidation. Proton extrusion remained unaltered when the proton-translocating ATPase was specifically inhibited by dicyclohexylcarbodiimide. The latter finding indicates that proton translocation is associated with CO conversion to CO2 and H2 rather than with ATP hydrolysis in the cells. The data substantiate that the coupling of CO oxidation with ADP phosphorylation in M. barkeri occurs via a chemiosmotic mechanism.

Archaea↗
PubMed1988

Citric-acid cycle, 50 years on. Modifications and an alternative pathway in anaerobic bacteria.

Many anaerobic bacteria can completely oxidize organic matter to CO2 with either sulfur, sulfate, or protons as electron acceptor. The sulfur-reducing bacteria and one genus of sulfate reducers use a modified citric-acid cycle with a novel anaplerotic sequence as pathway of terminal respiration. All other anaerobes use an alternative pathway, in which carbon monoxide dehydrogenase is a key enzyme and in which acetyl-CoA is cleaved into two C1 units at the oxidation level of CH3OH and CO. Thus almost 50 years after the discovery of the citric acid cycle by Hans Krebs in 1937, a second pathway for acetyl-CoA oxidation was found.

Acetates↗
PubMed1988

The final step in methane formation. Investigations with highly purified methyl-CoM reductase (component C) from Methanobacterium thermoautotrophicum (strain Marburg).

Methyl-coenzyme M reductase (= component C) from Methanobacterium thermoautotrophicum (strain Marburg) was highly purified via anaerobic fast protein liquid chromatography on columns of Mono Q and Superose 6. The enzyme was found to catalyze the reduction of methylcoenzyme M (CH3-S-CoM) with N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP = component B) to CH4. The mixed disulfide of H-S-CoM and H-S-HTP (CoM-S-S-HTP) was the other major product formed. The specific activity was up to 75 nmol min-1 mg protein-1. In the presence of dithiothreitol and of reduced corrinoids or titanium(III) citrate the specific rate of CH3-S-CoM reduction to CH4 with H-S-HTP increased to 0.5-2 mumol min-1 mg protein-1. Under these conditions the CoM-S-S-HTP formed from CH3-S-CoM and H-S-HTP was completely reduced to H-S-CoM and H-S-HTP. Methyl-CoM reductase was specific for H-S-HTP as electron donor. Neither N-6-mercaptohexanoylthreonine phosphate (H-S-HxoTP) nor N-8-mercaptooctanoylthreonine phosphate (H-S-OcoTP) nor any other thiol compound could substitute for H-S-HTP. On the contrary, H-S-HxoTP (apparent Ki = 0.1 microM) and H-S-OcoTP (apparent Ki = 15 microM) were found to be effective inhibitors of methyl-CoM reductase, inhibition being non-competitive with CH3-S-CoM and competitive with H-S-HTP.

Disulfides↗
PubMed1987

Biosynthesis of coenzyme F430 in methanogenic bacteria. Identification of 15,17(3)-seco-F430-17(3)-acid as an intermediate.

Coenzyme F430 is a hydroporphinoid nickel complex present in all methanogenic bacteria. It is part of the enzyme system which catalyzes methane formation from methyl-coenzyme M. We describe here that under certain conditions a second nickel porphinoid accumulates in methanogenic bacteria. The compound was identified at 15,17(3)-seco-F430-17(3)-acid. The structural assignment rests on 14C-labelling experiments, fast-atom-bombardment mass spectra, 1H-NMR spectra of the corresponding hexamethyl ester, and ultraviolet/visible spectral comparison with model compounds. In cell extracts and in intact cells of methanogenic bacteria, 15,17(3)-seco-F430-17(3)-acid was converted to F430. These findings indicate that the new nickel-containing porphinoid is an intermediate in the biosynthesis of coenzyme F430.

Coenzymes↗
PubMed1987

Proton-motive-force-driven formation of CO from CO2 and H2 in methanogenic bacteria.

Cell suspensions of methanogenic bacteria (Methanosarcina barkeri, Methanospirillum hungatei, Methano-brevibacter arboriphilus, and Methanobacterium thermoautotrophicum) were found to form CO from CO2 and H2 according to the reaction: CO2 + H2----CO + H2O; delta G0 = +20 kJ/mol. Up to 15,000 ppm CO in the gas phase were reached which is significantly higher than the equilibrium concentration calculated from delta G0 (95 ppm under the experimental conditions). This indicated that CO2 reduction with H2 to CO is energy-driven and indeed the cells only generated CO when forming CH4. The coupling of the two reactions was studied in more detail with acetate-grown cells of M. barkeri using methanogenic substrates. The effects of the protonophore tetrachlorosalicylanilide (TCS) and of the proton-translocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide (cHxN)2C were determined. TCS completely inhibited CO formation from CO2 and H2 without affecting methanogenesis from CH3OH and H2. In the presence of the protonophore the proton motive force delta p and the intracellular ATP concentration were very low. (cHxN)2C, which partially inhibited methanogenesis from CH3OH and H2, had no effect on CO2 reduction to CO. In the presence of (cHxN)2C delta p was high and the intracellular ATP content was low. These findings suggest that the endergonic formation of CO from CO2 and H2 is coupled to the exergonic formation of CH4 from CH3OH and H2 via the proton motive force and not via ATP. CO formation was not stimulated by the addition of sodium ions.

Adenosine Triphosphate↗
PubMed1987

On the role of N-7-mercaptoheptanoyl-O-phospho-L-threonine (component B) in the enzymatic reduction of methyl-coenzyme M to methane.

The reduction of methyl-coenzyme M (CH3SCoM) to methane in methanogenic bacteria is dependent on component B (N-7-mercaptoheptanoyl-O-phospho-L-threonine, HSHTP). We report here that S-methyl-component B (N-7-(methylthio)heptanoyl-O-phospho-L-threonine, CH3SHTP) can substitute for neither CH3SCoM nor HSHTP in the methyl-CoM reductase reaction. Rather, CH3SHTP proved to be an inhibitor competitive with HSHTP (apparent Ki = 6 microM) and noncompetitive with CH3SCoM. These results make it very unlikely that HSHTP functions as a methyl group carrier. A role for HSHTP as direct electron donor for CH3SCoM reduction to CH4 is proposed.

Chromatography, Gas↗
PubMed1987

7-Mercaptoheptanoylthreonine phosphate functions as component B in ATP-independent methane formation from methyl-CoM with reduced cobalamin as electron donor.

Purified methyl-CoM reductase of Methanobacterium thermoautotrophicum (strain Marburg) catalyzed the reduction of methyl-CoM to methane with reduced cobalamin, when either synthetic 7-mercaptoheptanoylthreonine phosphate (HS-HTP) or naturally occurring component B was present. With both compounds the same maximal specific activity was obtained and ATP was neither required nor stimulatory. These findings indicate that HS-HTP functions as component B and do not support the idea that HS-HT is only active in an adenosine monophosphorylated form.

Adenosine Triphosphate↗
PubMed1987

The corrinoid from Methanobacterium thermoautotrophicum (Marburg strain). Spectroscopic structure analysis and identification as Co beta-cyano-5'-hydroxybenzimidazolyl-cobamide (factor III).

The corrinoids from Methanobacterium thermoautotrophicum were extracted as the Co-cyano derivative, which was isolated in crystalline form. A consistent set of spectroscopic data was acquired (ultraviolet/visible, circular dichroic, infrared, fast-atom-bombardment mass, 1H-NMR and 13C-NMR spectra), which allowed the structural analysis of this complete corrinoid. It was assigned the structure of the Co beta-cyano-5'-hydroxybenzimidazolyl-cobamide and was identified with Friedrich and Bernhauer's 'factor III' by comparison with an authentic sample.

Circular Dichroism↗
PubMed1986

Coupling of carbon monoxide oxidation to CO2 and H2 with the phosphorylation of ADP in acetate-grown Methanosarcina barkeri.

Cell suspensions of Methanosarcina barkeri, grown on acetate, catalyzed the conversion of carbon monoxide and H2O to CO2 and H2 in stoichiometric amounts when methane formation was inhibited by bromoethanesulfonate. The specific activity was 80-120 nmol min-1 mg protein-1 at 5% CO in the gas phase. CO oxidation was coupled with the phosphorylation of ADP as indicated by a rapid increase of the intracellular ATP level upon start of the reaction. At least 0.1 mol ATP was formed/mol CO consumed. The onset of CO oxidation was also accompanied by an increase of the proton motive force (delta p) from 100 mV to 150 mV (inside negative). Addition of the uncoupler tetrachlorosalicylanilide to CO-metabolizing cells led to a rapid decrease of the ATP level and of delta p, and to an increase of the CO oxidation rate up to 70%. In the presence of the proton-translocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide the phosphorylation of ADP was inhibited and CO oxidation slowed down, whereas delta p was almost unaffected. Inhibition of CO oxidation under these conditions was relieved by the addition of the protonophore tetrachlorosalicylanilide. The results indicate that in acetate-grown M. barkeri the free-energy change associated with the formation of CO2 and H2 from CO and H2O (delta G degrees = -20 kJ/mol) can be used to drive the phosphorylation of ADP and that the coupling proceeds via a chemiosmotic mechanism. A possible role of the carbon monoxide oxidation reaction as an energy-conserving site in acetate fermentation to CH4 and CO2 is discussed.

Adenosine Diphosphate↗
PubMed1986

Methane formation from methyl-coenzyme M in a system containing methyl-coenzyme M reductase, component B and reduced cobalamin.

Purified methyl-CoM reductase of Methanobacterium thermoautotrophicum was found to catalyze the reduction of methyl-coenzyme M to methane at a specific rate of 100 nmol X min-1 X mg protein-1 in a system containing partially purified component B, cob(III)alamin (B-12a), and, as electron donors, dithiothreitol or SnCl2. Under these experimental conditions B-12a was reduced to cob(II)alamin (B-12r), which is known to disproportionate to cob(I)alamin (B-12s) and B-12a to a slight extent. Methane formation from methyl-CoM was inhibited by propyl iodide, methyl iodide, chloroform and carbon tetrachloride, which were shown to react with B-12s to the corresponding light-sensitive alkyl cobalamin. Inhibition by propyl iodide was less effective in light-exposed samples. From these findings it is concluded that in this assay system B-12s might serve as electron donor for the enzymatic reduction of methyl-CoM to methane.

Catalysis↗
PubMed1985

An extended-X-ray-absorption-fine-structure (e.x.a.f.s.) study of coenzyme F430 from Methanobacterium thermoautotrophicum.

Coenzyme F430 is a nickel porphinoid found in all methanogenic bacteria. Extended-X-ray-absorption-fine-structure (e.x.a.f.s.) spectra have been recorded above the nickel K-edge of coenzyme F430 and two model compounds, (5,10,15,20-tetramethylporphinato) nickel(II) and (5,10,15,20-tetramethylchlorinato)-nickel(II). The results show that the four nickel-nitrogen distances in F430 are split, with two nitrogen atoms at 0.192 nm and two at 0.210 nm.

Chemical Phenomena↗
PubMed1985

Is coenzyme M bound to factor F430 in methanogenic bacteria? Experiments with Methanobrevibacter ruminantium.

Coenzyme M (2-mercaptoethane sulfonic acid) and factor F430 (a nickel porphinoid) are coenzymes found in methanogenic bacteria. Recently it has been proposed that in these bacteria a coenzyme MF430 also exists which plays a key role in methane formation and in which coenzyme M and F430 are bound to each other. To test this hypothesis Methanobrevibacter ruminantium, which requires coenzyme M as a vitamin, was grown in the presence of [2-14C]CoMSH. F430 and 'CoM' (mixture of CoMSH and its disulfides) were quantitatively extracted from these cells and from partially purified methyl-CoM reductase using various methods. The extracts were chromatographed on cellulose or Sephadex G-10. Under all conditions factor F430 and 'CoM' were completely (greater than 99%) separated. There was no indication for the existence of a protein-free F430 species containing covalently bound coenzyme M in Mb. ruminantium. The results support the structure previously assigned to coenzyme F430.

Binding Sites↗
PubMed1985

Formation of carbon monoxide from CO2 and H2 by Methanobacterium thermoautotrophicum.

Cell suspensions of Methanobacterium thermoautotrophicum were found to reduce CO2 with H2 to CO at a maximal rate of 100 nmol X min-1 X mg protein-1. Half-maximal rates were obtained at a H2 and a CO2 concentration in the gas phase of 10% and 30%, respectively. The CO concentration in the gas phase surpassed the equilibrium concentration by a factor of more than 15 which indicates that CO2 reduction with H2 to CO was energy-driven. This was substantiated by the observation that the cells only formed CO when they also generated methane and that CO formation was completely inhibited by uncouplers. CO formation by cell suspensions and by growing cells was inhibited by cyanide. Neither methane formation nor the electrochemical proton potential were affected by this inhibitor. Cyanide was shown to inactivate specifically the carbon monoxide dehydrogenase present in M. thermoautotrophicum. It is therefore concluded that reduction of CO2 to CO is catalyzed by this enzyme. CO production by growing cells was 5-10-times slower than by resting cells. This is explained by effective CO assimilation in growing cells; when CO assimilation was inhibited by propyl iodide the rate of CO production immediately increased more than tenfold.

Aldehyde Oxidoreductases↗
PubMed1984

Studies on the biosynthesis of coenzyme F420 in methanogenic bacteria.

Coenzyme F420 is a 8-hydroxy-5-deazaflavin present in methanogenic bacteria. We have investigated whether the pyrimidine ring of the deazaflavin originates from guanine as in flavin biosynthesis, in which the pyrimidine ring of guanine is conserved. For this purpose the incorporation of [2-14C]guanine and of [8-14C]guanine into F420 by growing cultures of Methanobacterium thermoautotrophicum was studied. Only in the case of [2-14C]guanine did F420 become labeled. The specific radioactivity of the deazaflavin and of guanine isolated from nucleic acids of [2-14C]guanine grown cells were identical. This finding suggests that the pyrimidine ring of the deazaflavin and of flavins are synthesized by the same pathway. F420 did not become labeled when M. thermoautotrophicum was grown in the presence of methyl-[14C]methionine, [U-14C]phenylalanine or [U-14C]tyrosine. This excludes that C-5 of the deazaflavin is derived from the methyl group of methionine and that the benzene ring comes from phenylalanine or tyrosine.

Carbon Radioisotopes↗