An unusual thiol-driven fumarate reductase in Methanobacterium with the production of the heterodisulfide of coenzyme M and N-(7-mercaptoheptanoyl)threonine-O3-phosphate

An unusual fumarate reductase was purified from cell extracts of Methanobacterium thermoautotrophicum and partially characterized. Two coenzymes previously isolated from cell extracts, 2-mercaptoethane-sulfonic acid (HS-CoM) and N-(7-mercaptoheptanoyl)threonine-O3-phosphate (HS-HTP), were established as direct electron donors for fumarate reductase. By measuring the consumption of free thiol, we determined that fumarate reductase catalyzed the oxidation of HS-CoM and HS-HTP; by the direct measurement of succinate and the heterodisulfide of HS-CoM and HS-HTP (CoM-S-S-HTP), we established that these compounds were products of the fumarate reductase reaction. A number of thiol-containing compounds did not function as substrates for fumarate reductase, but this enzyme had high specific activity when HS-CoM and HS-HTP were used as electron donors. HS-CoM and HS-HTP were quantitatively oxidized by the fumarate reductase reaction, and results indicated that this reaction was irreversible. Additionally, by measuring formylmethanofuran, we demonstrated that the addition of fumarate to cell extracts activated CO2 fixation for the formation of formylmethanofuran. Results indicated that this activation resulted from the production of CoM-S-S-HTP (a compound known to be involved in the activation of formylmethanofuran synthesis) by the fumarate reductase reaction.     

Formyl-methanofuran synthesis in Methanobacterium thermoautotrophicum

The initial step of methanogenesis from CO2 is the formation of formyl-methanofuran (formyl-MFR) from methanofuran (MFR), CO2 and H2. The enzymology of this novel type of CO2 fixation reaction has been difficult to study because formyl-MFR synthesis is subject to a complex activation. Recently, however, a number of advances have made questions regarding formyl-MFR synthesis more approachable.     

Initial steps in the fermentation of 3-hydroxybenzoate by Sporotomaculum hydroxybenzoicum

The anaerobic bacterium Sporotomaculum hydroxybenzoicum ferments 3-hydroxybenzoate to acetate, butyrate, and CO2. 3-Hydroxybenzoate was activated to 3-hydroxybenzoyl-CoA in a CoA-transferase reaction with acetyl-CoA or butyryl-CoA as CoA donors. 3-Hydroxybenzoyl-CoA was reductively dehydroxylated, forming benzoyl-CoA. This reaction was measured in cell-free extracts with cob(I)alamin as low-potential electron donor. No evidence was obtained that cob(I)alamin is the physiological electron donor; however, inhibitor studies indicated involvement of a strong nucleophile in the reaction. Benzoate was degraded by dense cell suspensions without a lag phase until an in situ deltaG' value <-25 kJ mol(-1) was reached. Benzoyl-CoA reductase was not detected. Enzyme activities for all reaction steps from glutaryl-CoA to butyryl-CoA, and ATP formation via acetate kinase were detected in cell-free extracts. Glutaconyl-CoA decarboxylase is likely to act as a primary sodium ion pump.     

Formyl-methanofuran synthesis in Methanobacterium thermoautotrophicum

Abstract The initial step of methanogenesis from CO₂ is the formation of formyl-methanofuran (formyl-MFR) from methanofuran (MFR), CO₂ and H₂. The enzymology of this novel type of CO₂ fixation reaction has been difficult to study because formyl-MFR synthesis is subject to a complex activation. Recently, however, a number of advances have made questions regarding formyl-MFR synthesis more approachable.     

Coupling of methyl coenzyme M reduction with carbon dioxide activation in extracts of Methanobacterium thermoautotrophicum

The stimulation of carbon dioxide reduction to methane by addition of 2-(methylthio)ethanesulfonate (CH3-S-CoM) to cell extracts of Methanobacterium thermoautotrophicum was investigated. Similar stimulation of CO2 reduction by CH3-S-CoM was found for cell extracts of Methanobacterium bryantii and Methanospirillum hungatei. The CH3-S-CoM requirement could be met by the methanogenic precursors formaldehyde, serine, or pyruvate, or by 2-(ethylthio)ethanesulfonate (CH3CH2-S-CoM), but not by other coenzyme M derivatives. Efficient reduction of CO2 to CH4 was favored by low concentrations of CH3-S-CoM and high concentrations of CO2. Sulfhydryl compounds were identified as effective inhibitors of CO2 reduction. Both an allosteric model and a free-radical model for the mechanism of CO2 activation and reduction are discussed.     

Coenzyme F430 as a possible catalyst for the reductive dehalogenation of chlorinated C1 hydrocarbons in methanogenic bacteria

Corrinoids, such as aquocobalamin, methylcobalamin, and (cyanoaquo)cobinamide, catalyze the reductive dehalogenation of CCl4 with titanium(III) citrate as the electron donor [Krone et al. (1989) Biochemistry 28, 4908-4914]. We report here that this reaction is also effectively mediated by the nickel-containing porphinoid, coenzyme F430, found in methanogenic bacteria. Chloroform, methylene chloride, methyl chloride, and methane were detected as intermediates and products. Ethane was formed in trace amounts, and several as yet unidentified nonvolatile compounds were also generated. The rate of dehalogenation decreased in the series of CCl4, CHCl3, and CH2Cl2. With coenzyme F430 as the catalyst, the reduction of CH3Cl to CH4 proceeded more than 50 times faster than with aquocobalamin. Cell suspensions of Methanosarcina barkeri were found to catalyze the reductive dehalogenation of CCl4 with CO as the electron donor (E'0 = -0.524 V). Methylene chloride was the main end product. The kinetics of CHCl3 and CH2Cl2 formation from CCl4 were similar to those with coenzyme F430 or aquocobalamin as catalysts and titanium(III) citrate as the reductant.     

Stimulation of the methyltetrahydromethanopterin: coenzyme M methyltransferase reaction in cell-free extracts of Methanobacterium thermoautotrophicum by the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate

The conversion of formaldehyde to methylcoenzyme M in cell-free extracts of Methanobacterium thermoautotrophicum was stimulated up to 10-fold by catalytic amounts of the heterodisulfide (CoM-S-S-HTP) of coenzyme M and 7-mercaptoheptanoylthreonine phosphate. The stimulation required the additional presence of ATP, also in catalytic concentrations. ATP and CoM-S-S-HTP were mutually stimulatory on the methylcoenzyme M formation and it was concluded that the compounds were both involved in the reductive activation of the methyltetrahydromethanopterin: coenzyme M methyltransferase. Micromolar concentrations of benzyl viologen or cyanocobalamin inhibited the formaldehyde conversion; these compounds, however, strongly stimulated the reduction of CoM-S-S-HTP. The results described here closely resemble observations made on the activation and reduction of CO₂ to formylmethanofuran indicating that this step and the reductive activation of the methyltransferase are controlled by some common mechanism.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme M, 2-(methylthio)ethanesulfonate - H₄MPT 5,6,7,8-tetrahydromethanopterin - MFR methanofuran - HS-HTP 7-mercaptoheptanoylthreonine phosphate - CoM-S-S-HTP the heterodisulfide of HS-CoM and HS-HTP - BES 2-bromoethanesulfonate - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - CN-Cbl cyanocobalamin - HO-Cbl hydroxycobalamin - HBI 5-hydroxybenzimidazole - DMBI 5,6-dimethylbenzimidazole     

Dissimilatory sulphate reduction with acetate as electron donor

Acetate oxidation by sulphate was studied with desulfobacter postgatei. Cell extracts of the organism were found to contain high activities of the following enzymes: citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase and pyruvate synthase. It is concluded that acetate oxidation with sulphate in D. postgatei proceeds via the citric acid cycle with the synthesis of pyruvate from acetyl CoA and CO2 as an anaplerotic reaction. The apparent Ks for acetate oxidation by D. postgatei as determined in vivo was near 0.2 mM. The apparent Ks for acetate fermentation to methane and CO2 by methanosarcina barkeri was 3 mM. The significantly lower ks for acetate of the sulphate reducer explains why methane formation from acetate in natural habitats is apparently inhibited by sulphate.     

The biosynthesis of valine from isobutyrate by Peptostreptococcus elsdenii and Bacteroides ruminicola

1. Growing cultures of Peptostreptococcus elsdenii and Bacteroides ruminicola incorporate (14)C from [1-(14)C]isobutyrate into the valine of cell protein. With P. elsdenii some of the (14)C is also incorporated into leucine. 2. Crude cell-free extracts of both organisms in the presence of glutamine, carbon dioxide and suitable sources of energy and electrons incorporate (14)C from [1-(14)C]isobutyrate into valine but not into leucine. 3. With extracts of P. elsdenii treated with DEAE-cellulose the reaction is dependent on ATP, CoA, thiamin pyrophosphate, molecular hydrogen and a low-potential electron carrier (ferredoxin, flavodoxin or benzyl viologen). 4. The same extracts incorporate (14)C from NaH(14)CO(3) into valine in the presence of isobutyrate plus ATP, CoA, glutamine and ferredoxin; isobutyryl-CoA or isobutyryl phosphate plus CoA will replace the isobutyrate plus CoA and ATP. With acetyl phosphate in place of isobutyryl phosphate, (14)C is incorporated into alanine. With isovalerate or 2-methylbutyrate in place of isobutyrate, (14)C is incorporated into leucine and isoleucine respectively. 5. When carrier 2-oxoisovalerate is added to the carboxylating system (14)C from [1-(14)C]isobutyrate passes into the oxo acid fraction. 6. It is concluded that these two organisms form valine from isobutyrate by the sequence isobutyrate-->isobutyryl-CoA-->2-oxoisovalerate-->valine and that the reductive carboxylation of isobutyrate is catalysed by a system similar to the pyruvate synthetase of clostridia and photosynthetic bacteria.     

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.     

Evidence that the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate is a product of the methylreductase reaction in Methanobacterium

When 7-mercaptoheptanoylthreonine phosphate (HS-HTP) was used as the sole source of electrons for reductive demethylation of 2-(methylthio)-ethanesulfonic acid (CH3-S-CoM) by cell extracts of Methanobacterium thermoautotrophicum strain delta H, the heterodisulfide of coenzyme M and HS-HTP (CoM-S-S-HTP) was quantitatively produced: HS-HTP + CH3-S-CoM----CH4 + CoM-S-S-HTP. CH4 and CoM-S-S-HTP were produced stoichiometrically in a ratio of 1:1. Coenzyme M (HS-CoM) inhibited HS-HTP driven methanogenesis indicating that CH3-S-CoM rather than HS-CoM was the substrate for CoM-S-S-HTP formation.     

Methanogenesis from acetate in cell extracts of Methanosarcina barkeri: Isotope exchange between CO2 and the carbonyl group of acetyl-CoA,and the role of H2

From our previous studies on the mechanism of methane formation from acetate it was known that cell extracts of acetate-grown Methanosarcina barkeri (100 000 × g supernatant) catalyze the conversion of acetyl-CoA plus tetrahydromethanopterin (=H₄MPT) to methyl-H₄MPT, CoA, CO₂ and presumably H₂. We report here that these extracts, in the absence of H₄MPT, mediated an isotope exchange between CO₂ ([S]_{0.5 v}=0.2% in the gas phase) and the carbonyl group of acetyl-CoA at almost the same specific rate as the above conversion (10 nmol · min^–1 · mg protein^–1). Both the exchange and the formation of methyl-H₄MPT were inhibited by N₂O, suggesting that a corrinoid could be the primary methyl group acceptor in the acetyl-CoA C-C-cleavage reaction. Both activities were dependent on the presence of H₂ (E⁰=–414 mV). Ti(III)citrate (E⁰=–480 mV) was found to substitute for H₂, indicating a reductive activation of the system. In the presence of Ti(III)citrate it was shown that the formation of CO₂ from the carbonyl group of acetyl-CoA is associated with a 1:1 stoichiometric generation of H₂. Free CO, a possible intermediate in CO₂ and H₂ formation, was not detected.Non-standard abbreviations AcCoA acetyl-CoA - acetyl-P acetyl phosphate - OH-B₁₂ hydroxocobalamin - H-S-CoM coenzyme M = 2-mercaptoethanesulfonate - CH₃-S-CoM methyl-coenzyme M = 2-(methylthio)ethanesulfonate - H-S-HTP N-7-mercaptoheptanoylthreonine phosphate - HTP-S-S-HTP disulfide of H-S-HTP - CoM-S-S-HTP disulfide of H-S-CoM and H-S-HTP - H₄MPT tetrahydromethanopterin - CH₃-H₄MPT N⁵-methyl-H₄MPT - DTT dithiothreitol - MOPS morpholinopropane sulfonic acid     

Ferredoxin-dependent methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS).

Cell extracts of Methanosarcina barkeri grown on acetate catalyzed the conversion of acetyl-CoA to CO2 and CH4 at a specific rate of 50 nmol min-1 mg-1. When ferredoxin was removed from the extracts by DEAE-Sephacel anion exchange chromatography, the extracts were inactive but full activity was restored upon addition of purified ferredoxin from M. barkeri or from Clostridium pasteurianum. The apparent Km for ferredoxin from M. barkeri was determined to be 2.5 M. A ferredoxin dependence was also found for the formation of CO2, H2 and methylcoenzyme M from acetyl-CoA, when methane formation was inhibited by bromoethanesulfonate. Reduction of methyl-coenzyme M with H2 did not require ferredoxin. These and other data indicate that ferredoxin is involved as electron carrier in methanogenesis from acetate. Methanogenesis from acetyl-CoA in cell extracts was not dependent on the membrane fraction, which contains the cytochromes.     

Citrate and the conversion of carbohydrate into fat. The regulation of fatty acid synthesis by rat liver extracts

1. The rate of fatty acid synthesis by particle-free extracts prepared from rat liver is increased greatly if the enzyme system is first activated with citrate. 2. The extent of the activation depends on the citrate concentration and on the time of activation in an interdependent manner. 3. Citrate activation is strongly dependent on temperature. 4. Tricarballylate can replace citrate as an activator, but its presence in the assay inhibits fatty acid synthesis. 5. Mg(2+) ions can replace citrate in the activation but not in the complete reaction system. 6. ATP prevents the activating effect of citrate and Mg(2+) ions. 7. The rate of fatty acid synthesis is increased by palmitoyl-dl-carnitine. This type of activation, additional to that caused by citrate, is rapid and does not depend on prior incubation. 8. Inhibition of fatty acid synthesis by palmitoyl-CoA can be prevented by palmitoyl-dl-carnitine or by increasing the concentration of protein.     

Nitrogenase Activity in Cell-Free Extracts of the Blue-Green Alga, Anabaena cylindrica

Cell-free extracts with high nitrogenase activity were prepared by sonic oscillation and French press treatment from the blue-gree alga Anabaena cylindrica. Extracts were prepared from cells grown on a 95% N(2)-5% CO(2) gas mixture followed by a period of nitrogen starvation under an atmosphere of 95% argon-5% CO(2). No increase in the specific activity of extracts was achieved by breaking heterocysts. Activity (assayed by acetylene reduction) was found to be dependent on adenosine triphosphate (ATP), an ATP-generating system, and a low-potential reductant. Na(2)S(2)O(2) employed as reductant supports higher rates of nitrogenase activity than reduced ferredoxin. The activity is associated with a small-particle fraction that can be sedimented by ultracentrifugation. In contrast to the particulate nitrogenase of Azotobacter, which is stable in air, the A. cylindrica nitrogenase is an oxygen sensitive as nitrogenase prepared from anaerobic bacteria.     

Structure and function of a menaquinone involved in electron transport in membranes of Clostridium thermoautotrophicum and Clostridium thermoaceticum.

Clostridium thermoaceticum and Clostridium thermoautotrophicum contain the same menaquinone. Its structure, determined by thin-layer chromatography, UV absorption spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy, was found to be MK-7 (2-methyl-3-heptaprenyl-1,4-naphthoquinone). The menaquinone is located in the cytoplasmic membranes and is involved in redox reactions of two b-type cytochromes present in the clostridia. These reactions were studied with right-side-out membranes prepared from C. thermoautotrophicum by using CO as an electron donor. In intact membranes, both cytochromes were reduced, whereas after inactivation of the menaquinone by exposure of the membranes to UV irradiation, reduction of the low-potential cytochrome (Eo', -200 mV) but not of the high-potential cytochrome (Eo', -48 mV) occurred. The reduction of the high-potential cytochrome in UV-irradiated membranes was restored following the addition of oxidized menaquinone and with an excess of CO. The addition of oxidized menaquinone to reduced membranes resulted initially in a preferential oxidation of the low-potential cytochrome. The results obtained indicate that the menaquinone acts between the two b-type cytochromes in an electron transport chain.     

ATP synthesis coupled to methane formation from methyl-CoM and H2 catalyzed by vesicles of the methanogenic bacterial strain G?1

Methanogenesis from methyl-CoM and H2, as catalyzed by inside-out vesicle preparations of the methanogenenic bacterium strain G?1, was associated with ATP synthesis. That this ATP synthesis proceeded via an uncoupler-sensitive transmembrane proton gradient was concluded from the following results: 1. Various inhibitors that affected methane formation (e.g. 2-bromomethanesulfonate) also prevented ATP synthesis. 2. The protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile, in combination with the K+ ionophore valinomycin, inhibited ATP synthesis completely without affecting methanogenesis. 3. The ATP synthase inhibitor diethylstilbestrol inhibited ATP synthesis. 4. Addition of the detergent sulfobetaine inhibited both methane formation and ATP synthesis; the former but not the latter could be restored by adding titanium(III) citrate as electron donor. In addition it was shown that ATP synthesis could also be driven by transmembrane proton gradients artificially imposed on the vesicles. Furthermore net methanogenesis-dependent ATP formation was shown by measuring [32P]phosphate incorporation.     

Sodium ions and an energized membrane required by Methanosarcina barkeri for the oxidation of methanol to the level of formaldehyde.

Methanogenesis from methanol by cell suspensions of Methanosarcina barkeri was inhibited by the uncoupler tetrachlorosalicylanilide. This inhibition was reversed by the addition of formaldehyde. 14C labeling experiments revealed that methanol served exclusively as the electron acceptor, whereas formaldehyde was mainly oxidized to CO2 under these conditions. These data support the hypothesis (M. Blaut and G. Gottschalk, Eur. J. Biochem. 141: 217-222, 1984) that the first step in methanol oxidation depends on the proton motive force or a product thereof. Cell extracts of M. barkeri converted methanol and formaldehyde to methane under an H2 atmosphere. Under an N2 atmosphere, however, formaldehyde was disproportionated to CH4 and CO2, whereas methanol was metabolized to a very small extent only, irrespective of the presence of ATP. It was concluded that cell extracts of M. barkeri are not able to oxidize methanol. In further experiments, the sodium dependence of methanogenesis and ATP formation by whole cells was investigated. Methane formation from methanol alone and the corresponding increase in the intracellular ATP content were strictly dependent on Na+. If, in contrast, methanol was utilized together with H2, methane and ATP were synthesized in the absence of Na+. The same is true for the disproportionation of formaldehyde to methane and carbon dioxide. From these experiments, it is concluded that in M. barkeri, Na+ is involved not in the process of ATP synthesis but in the first step of methanol oxidation.     

Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis.

To detect autotrophic CO2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotrophically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO2 fixation was pulled in the direction of lactate synthesis, CO2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO2 and H2, but H2 + CO2-independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min-1 mg of protein-1. When BES was added, the rate of lactate synthesis increased to 2.3 nmol min-1 mg of protein-1. Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14CO2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14CH2O was specifically incorporated into the C-3 of lactate, and 14CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO2 assimilation.     

Quinones as hydrogen carriers for a late step in anaerobic heme biosynthesis in Escherichia coli

A late step in anaerobic heme synthesis, the oxidation of protoporphyrinogen with fumarate as electron acceptor, was studied in extracts and particles of Escherichia coli mutants deficient in quinones or cytochromes. Mutants specifically deficient in menaquinone did not couple protoporphyrinogen oxidation to fumarate reduction, whereas mutants containing menaquinone but deficient in either ubiquinone or cytochromes exhibited this activity. These findings indicate that this coupled reaction is dependent upon menaquinone as hydrogen carrier but independent of ubiquinone and cytochromes. Other characteristics of this coupled reaction were also studied. The activity was located exclusively in the membrane fraction of cell-free extracts. Coproporphyrinogen III could not replace protoporphyrinogen as substrate. Methylene blue, triphenyl tetrazolium and nitrate, but not nitrite, could replace fumarate as anaerobic hydrogen acceptor. These findings have implications for the mechanism and regulation of microbial heme and chlorophyll synthesis and for the physiology of cytochrome synthesis in anaerobic microorganisms.