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Rudolf K Thauer - One of the best experts on this subject based on the ideXlab platform.

  • heme biosynthesis in Methanosarcina barkeri via a pathway involving two methylation reactions
    Journal of Bacteriology, 2006
    Co-Authors: Barbel Buchenau, Jorg Kahnt, Ilka U Heinemann, Dieter Jahn, Rudolf K Thauer
    Abstract:

    The methanogenic archaeon Methanosarcina barkeri synthesizes protoheme via precorrin-2, which is formed from uroporphyrinogen III in two consecutive methylation reactions utilizing S-adenosyl-l-methionine. The existence of this pathway, previously exclusively found in the sulfate-reducing δ-proteobacterium Desulfovibrio vulgaris, was demonstrated for M. barkeri via the incorporation of two methyl groups from methionine into protoheme.

  • protection of Methanosarcina barkeri against oxidative stress identification and characterization of an iron superoxide dismutase
    Archives of Microbiology, 2000
    Co-Authors: Andrei L Brioukhanov, Rudolf K Thauer, A I Netrusov, Melanie Sordel, Seigo Shima
    Abstract:

    Methanosarcina barkeri is a methanogenic archaeon that can only grow under strictly anoxic conditions but which can survive oxidative stress. We have recently reported that the organism contains a monofunctional catalase. We describe here that it also possesses an active iron superoxide dismutase. The enzyme was purified in three steps over 130-fold in a 14% yield to a specific activity of 1500 U/mg. SDS-PAGE revealed the presence of only one band, at an apparent molecular mass of 25 kDa. The primary structure determined from the cloned and sequenced gene revealed similarity to iron- and manganese superoxide dismutases. The highest similarity was to the iron superoxide dismutase from Methanobacterium thermoautotrophicum. The enzyme from M. barkeri was found to contain, per mol, 1 mol iron, but no manganese – in agreement with the general observation that anaerobically growing organisms only contain iron superoxide dismutase. The enzyme was not inhibited by cyanide (10 mM), which is a property shared by all iron- and manganese superoxide dismutases. The presence of superoxide dismutase in M. barkeri is noteworthy since a gene encoding superoxide dismutase (sod) has not been found in Archaeoglobus fulgidus, a sulfate-reducing archaeon most closely related to the Methanosarcinaceae.

  • purification characterization and primary structure of a monofunctional catalase from Methanosarcina barkeri
    Archives of Microbiology, 1999
    Co-Authors: Seigo Shima, A I Netrusov, Melanie Sordel, Michaela Wicke, Gudrun C Hartmann, Rudolf K Thauer
    Abstract:

    Methanosarcina barkeri is a strictly anaerobic, cytochrome-containing, methane-forming archaeon. We report here that the microorganism contains a catalase, which was purified and characterized. The enzyme with an apparent molecular mass of 190 kDa was shown to be composed of four identical subunits of apparent molecular mass of 54 kDa. The heme-containing enzyme did not exhibit peroxidase activity, which indicates that it is a monofunctional catalase. This is substantiated by the primary structure, which is related to that of other monofunctional catalases rather than to that of bifunctional catalase-peroxidases. The enzyme showed an [S]0.5V for H2O2 of 25 mM and an apparent Vmax of 200,000 U/mg; it was inhibited by azide ([I]0.5V = 1 μM) and cyanide ([I]0.5V = 5 μM) and inactivated by 1,2,4-aminotriazole. The activity was almost independent of the pH (between pH 4 and 10) and the temperature (between 15 °C and 55 °C). Comparison of the primary structure of monofunctional catalases revealed that the enzyme from M. barkeri is most closely related to the monofunctional catalase of Dictyostelium discoideum.

  • Two F420-reducing hydrogenases in Methanosarcina barkeri
    Archives of Microbiology, 1998
    Co-Authors: Martin Vaupel, Rudolf K Thauer
    Abstract:

    F420-reducing hydrogenases are nickel iron-sulfur flavoproteins involved in CO2 reduction with H2 to methane in methanogenic archaea. Evidence is presented that Methanosarcina barkeri contains two isoenzymes for which the encoding genes have been cloned and sequenced. The genes are organized in two operons, frhADGB and freAEGB, each comprising four open reading frames. Transcription analysis revealed that both operons are transcribed during growth of Ms. barkeri on H2/CO2, on methanol, and on trimethylamine, but not during growth on acetate.

  • methanol coenzyme m methyltransferase from Methanosarcina barkeri zinc dependence and thermodynamics of the methanol cob i alamin methyltransferase reaction
    FEBS Journal, 1997
    Co-Authors: Karin Sauer, Rudolf K Thauer
    Abstract:

    In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methyl-coenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated methyltransferases 1 (MT1) and 2 (MT2). Transferase MT1, which is composed of a 50-kDa subunit, MtaB, and a 27-kDa corrinoid-harbouring subunit, MtaC, has been shown recently to catalyze the methylation of free cob(I)alamin with methanol [Sauer, K., Harms, U. & Thauer, R. K. (1997) Eur. J. Biochem. 243, 670-677]. We report here that this reaction is catalyzed by subunit MtaB overproduced in Escherichia coli. MtaB also catalyzed the formation of methanol from methylcobalamin and H2O, the hydrolysis being associated with a free-energy change deltaG(o)' of approximately +7.0 kJ/mol. MtaB was found to contain 1 mol zinc, and its activity to be zinc dependent (pK(Zn2+) = 9.3). The zinc dependence of the MT2 (MtaA)-catalyzed reaction is also described (pK(Zn2+) = 9.6).

William W Metcalf - One of the best experts on this subject based on the ideXlab platform.

  • energy conservation via hydrogen cycling in the methanogenic archaeon Methanosarcina barkeri
    Mbio, 2018
    Co-Authors: Gargi Kulkarni, Thomas D. Mand, William W Metcalf
    Abstract:

    ABSTRACT Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H 2 production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon Methanosarcina barkeri is capable of energy conservation via H 2 cycling, based on genetic data that suggest that H 2 is a preferred, but nonessential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H 2 cycling. M. barkeri produces H 2 during growth on methanol, a phenotype that is lost upon mutation of the cytoplasmic hydrogenase encoded by frhADGB , although low levels of H 2 , attributable to the Ech hydrogenase, accumulate during stationary phase. In contrast, mutations that conditionally inactivate the extracellular Vht hydrogenase are lethal when expression of the vhtGACD operon is repressed. Under these conditions, H 2 accumulates, with concomitant cessation of methane production and subsequent cell lysis, suggesting that the inability to recapture extracellular H 2 is responsible for the lethal phenotype. Consistent with this interpretation, double mutants that lack both Vht and Frh are viable. Thus, when intracellular hydrogen production is abrogated, loss of extracellular H 2 consumption is no longer lethal. The common occurrence of both intracellular and extracellular hydrogenases in anaerobic microorganisms suggests that this unusual mechanism of energy conservation may be widespread in nature. IMPORTANCE ATP is required by all living organisms to facilitate essential endergonic reactions required for growth and maintenance. Although synthesis of ATP by substrate-level phosphorylation is widespread and significant, most ATP is made via the enzyme ATP synthase, which is energized by transmembrane chemiosmotic gradients. Therefore, establishing this gradient across the membrane is of central importance to sustaining life. Experimental validation of H 2 cycling adds to a short list of mechanisms for generating a transmembrane electrochemical gradient that is likely to be widespread, especially among anaerobic microorganisms.

  • energy conservation via hydrogen cycling in the methanogenic archaeon Methanosarcina barkeri
    bioRxiv, 2018
    Co-Authors: Gargi Kulkarni, Thomas D. Mand, William W Metcalf
    Abstract:

    Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H2 production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon Methanosarcina barkeri is capable of energy conservation via H2 cycling, based on genetic data that suggest H2 is a preferred, but non-essential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H2 cycling. M. barkeri produces H2 during growth on methanol, a phenotype that is lost upon mutation of the cytoplasmic hydrogenase encoded by frhADGB, although low levels of H2, attributable to the Ech hydrogenase, accumulate during stationary phase. In contrast, mutations that conditionally inactivate the extracellular Vht hydrogenase are lethal when expression of the vhtGACD operon is repressed. Under these conditions H2 accumulates, with concomitant cessation of methane production and subsequent cell lysis, suggesting that the inability to recapture extracellular H2 is responsible for the lethal phenotype. Consistent with this interpretation, double mutants that lack both Vht and Frh are viable. Thus, when intracellular hydrogen production is abrogated, loss of extracellular H2 consumption is no longer lethal. The common occurrence of both intracellular and extracellular hydrogenases in anaerobic microorganisms suggests that this unusual mechanism of energy conservation may be widespread in nature. Importance: Adenosine triphosphate (ATP) is required by all living organisms to facilitate essential endergonic reactions required for growth and maintenance. Although synthesis of ATP by substrate-level phosphorylation is widespread and significant, most ATP is made via the enzyme ATP synthase, which is energized by transmembrane chemiosmotic gradients. Therefore, establishing this gradient across the membrane is of central importance to sustaining life. Experimental validation of H2 cycling adds to a short list of mechanisms for generating a transmembrane electrochemical gradient that is likely to be widespread, especially among anaerobic microorganisms.

  • genetic biochemical and molecular characterization of Methanosarcina barkeri mutants lacking three distinct classes of hydrogenase
    bioRxiv, 2018
    Co-Authors: Thomas D. Mand, Gargi Kulkarni, William W Metcalf
    Abstract:

    ABSTRACT The methanogenic archaeon Methanosarcina barkeri encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F420-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H2/CO2, whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to wild-type, produced methane and CO2 in the expected 3:1 ratio, but had a ca. 33% slower growth rate. Thus, hydrogenases play a significant, but non-essential, role during growth on this substrate. As previously observed, mutants lacking Ech fail to grow on methanol/H2 unless supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the Δech/Δfrh and Δech/Δfrh/Δvht mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H2, which prevents production of reducing equivalents needed for biosynthesis. Quantification of methane and CO2 produced from methanol by resting cell suspensions of various mutants supports this conclusion. Based on global transcriptional profiles, none of the hydrogenases are upregulated to compensate for loss of the others. However, transcript levels of the F420 dehydrogenase operon were significantly higher in all strains lacking frh, suggesting a mechanism to sense the redox state of F420. The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed.

  • Energy Conservation via Hydrogen Cycling in the Methanogenic Archaeon Methanosarcina barkeri
    American Society for Microbiology, 2018
    Co-Authors: Gargi Kulkarni, Thomas D. Mand, William W Metcalf
    Abstract:

    Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H2 production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon Methanosarcina barkeri is capable of energy conservation via H2 cycling, based on genetic data that suggest that H2 is a preferred, but nonessential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H2 cycling. M. barkeri produces H2 during growth on methanol, a phenotype that is lost upon mutation of the cytoplasmic hydrogenase encoded by frhADGB, although low levels of H2, attributable to the Ech hydrogenase, accumulate during stationary phase. In contrast, mutations that conditionally inactivate the extracellular Vht hydrogenase are lethal when expression of the vhtGACD operon is repressed. Under these conditions, H2 accumulates, with concomitant cessation of methane production and subsequent cell lysis, suggesting that the inability to recapture extracellular H2 is responsible for the lethal phenotype. Consistent with this interpretation, double mutants that lack both Vht and Frh are viable. Thus, when intracellular hydrogen production is abrogated, loss of extracellular H2 consumption is no longer lethal. The common occurrence of both intracellular and extracellular hydrogenases in anaerobic microorganisms suggests that this unusual mechanism of energy conservation may be widespread in nature.ATP is required by all living organisms to facilitate essential endergonic reactions required for growth and maintenance. Although synthesis of ATP by substrate-level phosphorylation is widespread and significant, most ATP is made via the enzyme ATP synthase, which is energized by transmembrane chemiosmotic gradients. Therefore, establishing this gradient across the membrane is of central importance to sustaining life. Experimental validation of H2 cycling adds to a short list of mechanisms for generating a transmembrane electrochemical gradient that is likely to be widespread, especially among anaerobic microorganisms

  • Genetic, Genomic, and Transcriptomic Studies of Pyruvate Metabolism in Methanosarcina barkeri Fusaro
    Journal of bacteriology, 2015
    Co-Authors: Madeline M. López Muñoz, Peter Schönheit, William W Metcalf
    Abstract:

    UNLABELLED Pyruvate, a central intermediate in the carbon fixation pathway of methanogenic archaea, is rarely used as an energy source by these organisms. The sole exception to this rule is a genetically uncharacterized Methanosarcina barkeri mutant capable of using pyruvate as a sole energy and carbon source (the Pyr(+) phenotype). Here, we provide evidence that suggests that the Pyr(+) mutant is able to metabolize pyruvate by overexpressing pyruvate ferredoxin oxidoreductase (por) and mutating genes involved in central carbon metabolism. Genomic analysis showed that the Pyr(+) strain has two mutations localized to Mbar_A1588, the biotin protein ligase subunit of the pyruvate carboxylase (pyc) operon, and Mbar_A2165, a putative transcriptional regulator. Mutants expressing the Mbar_A1588 mutation showed no growth defect compared to the wild type (WT), yet the strains lacked pyc activity. Recreation of the Mbar_A2165 mutation resulted in a 2-fold increase of Por activity and gene expression, suggesting a role in por transcriptional regulation. Further transcriptomic analysis revealed that Pyr(+) strains also overexpress the gene encoding phosphoenolpyruvate carboxylase, indicating the presence of a previously uncharacterized route for synthesizing oxaloacetate in M. barkeri and explaining the unimpaired growth in the absence of Pyc. Surprisingly, stringent repression of the por operon was lethal, even when the media were supplemented with pyruvate and/or Casamino Acids, suggesting that por plays an unidentified essential function in M. barkeri. IMPORTANCE The work presented here reveals a complex interaction between anabolic and catabolic pathways involving pyruvate metabolism in Methanosarcina barkeri Fusaro. Among the unexpected findings were an essential role for the enzyme pyruvate-ferredoxin oxidoreductase and an alternate pathway for synthesis of oxaloacetate. These results clarify the mechanism of methanogenic catabolism of pyruvate and expand our understanding of carbon assimilation in methanogens.

Shungui Zhou - One of the best experts on this subject based on the ideXlab platform.

  • light driven carbon dioxide reduction to methane by Methanosarcina barkeri in an electric syntrophic coculture
    The ISME Journal, 2021
    Co-Authors: Lingyan Huang, Shungui Zhou, Xing Liu, Zhishuai Zhang, Christopher Rensing, Kenneth H Nealson
    Abstract:

    The direct conversion of CO2 to value-added chemical commodities, thereby storing solar energy, offers a promising option for alleviating both the current energy crisis and global warming. Semiconductor-biological hybrid systems are novel approaches. However, the inherent defects of photocorrosion, photodegradation, and the toxicity of the semiconductor limit the application of these biohybrid systems. We report here that Rhodopseudomonas palustris was able to directly act as a living photosensitizer to drive CO2 to CH4 conversion by Methanosarcina barkeri under illumination after coculturing. Specifically, R. palustris formed a direct electric syntrophic coculture with M. barkeri. Here, R. palustris harvested solar energy, performed anoxygenic photosynthesis using sodium thiosulfate as an electron donor, and transferred electrons extracellularly to M. barkeri to drive methane generation. The methanogenesis of M. barkeri in coculture was a light-dependent process with a production rate of 4.73 ± 0.23 μM/h under light, which is slightly higher than that of typical semiconductor-biohybrid systems (approximately 4.36 μM/h). Mechanistic and transcriptomic analyses showed that electrons were transferred either directly or indirectly (via electron shuttles), subsequently driving CH4 production. Our study suggests that R. palustris acts as a natural photosensitizer that, in coculture with M. barkeri, results in a new way to harvest solar energy that could potentially replace semiconductors in biohybrid systems.

  • electricity from anaerobic methane oxidation by a single methanogenic archaeon Methanosarcina barkeri
    Chemical Engineering Journal, 2021
    Co-Authors: Eryi Zhang, Zhiyong Jason Ren, Shungui Zhou
    Abstract:

    Abstract Anaerobic oxidation of methane (AOM) coupled with electron transfer to electrodes has recently showed great promise for energy harvesting in microbial electrochemical reactors. To date this process has been reported limited to the syntrophic cooperation of archaea and bacteria in mixed cultures. It remains unknown whether pure archaea alone can catalyze electricity production from methane. Here we report the performance of AOM-driven electricity generation by a single methanogen Methanosarcina barkeri. At a poised electrode potential of +300 mV vs. SHE, a maximum current of 649.7 mA m−2 was achieved from methane with an appreciable Coulombic efficiency up to 86.9%. The electrogenic mechanism analysis demonstrated that M. barkeri donated most of electrons to electrodes directly and secreted soluble redox-active compounds to facilitate indirect extracellular electron transfer (EET). Electrochemical in situ Fourier transform infrared spectra and metabolic inhibitions suggested the methyl-coenzyme M reductase α-subunit (McrA), NiFe hydrogenases and cytochrome proteins were involved in the AOM-EET process. This work provides the first proof of concept for AOM-powered electricity production by a single archaeon via reversed methanogenesis and suggests methanogens may play an important role in coupling AOM with the reduction of solid electron acceptors in natural environments.

  • efficient photoelectron capture by ni decoration in Methanosarcina barkeri cds biohybrids for enhanced photocatalytic co2 to ch4 conversion
    iScience, 2020
    Co-Authors: Jie Ye, Li Kang, Yiyun Zhang, Shungui Zhou, Zhen He
    Abstract:

    Summary Semi-artificial photosynthesis (biohybrid) provides an intriguing opportunity for efficient CO2-to-CH4 conversion. However, creating a desirable semiconductor in biohybrids remains a great challenge. Here, by doping Ni into CdS nanoparticles, we have successfully developed the Methanosarcina barkeri-Ni:CdS biohybrids. The CH4 yield by the M. barkeri-Ni(0.75%):CdS biohybrids was approximately 250% higher than that by the M. barkeri-CdS biohybrids. The suitable Ni dopants serve as an effective electron sink, which accelerates the photoelectron transfer in biohybrids. In addition, Ni doping changes the metabolic status of M. barkeri and results in a higher expression of a series of proteins for electron transfer, energy conversion, and CO2 fixation. These increased proteins can promote the photoelectron capture by M. barkeri and injection into cells, which trigger a higher intracellular reduction potential to drive the reduction of CO2 to CH4. Our discovery will offer a promising strategy for the optimization of biohybrids in the solar-to-chemical conversion.

  • light driven carbon dioxide reduction to methane by Methanosarcina barkeri cds biohybrid
    Applied Catalysis B-environmental, 2019
    Co-Authors: Yiyun Zhang, Man Chen, Xing Liu, Shungui Zhou
    Abstract:

    Abstract Semi-artificial photosynthesis has emerged as a promising approach to convert carbon dioxide to value-added chemicals. Herein, direct CO2-to-CH4 conversion was realized by an innovative biohybrid consisting of semiconductor nanoparticles and non-phototrophic methanogens. The interaction between a model methanogen Methanosarcina barkeri and photoactive CdS nanoparticles achieved a CH4 production rate of 0.19 μmol/h with a quantum efficiency of 0.34%, comparable to that of plants or algae. The M. barkeri-CdS biohybrid exhibited a higher electrical conductivity than M. barkeri only and generated photocurrent in response to irradiation. The simultaneous increase of mcrA gene copies by 151.4% illustrated the robustness of this M. barkeri-CdS biohybrid. Membrane-bound proteins were found to play a key role in the photoelectron transfer. The CO2-to-CH4 conversion was possibly conducted with photoelectrons from the e−-h+ separation via the H2ases-mediated and cytochromes-mediated pathways. The findings encourage further exploration of the solar-driven self-replicating biocatalytic system to achieve CO2-to-CH4 conversion.

  • nanofe3o4 as solid electron shuttles to accelerate acetotrophic methanogenesis by Methanosarcina barkeri
    Frontiers in Microbiology, 2019
    Co-Authors: Jingyuan Wang, Li Fu, Ting Zhou, Yahai Lu, Linpeng Yu, Shungui Zhou
    Abstract:

    Magnetite nanoparticles (nanoFe3O4) have been reported to facilitate direct interspecies electron transfer (DIET) between syntrophic bacteria and methanogens thereby improving syntrophic methanogenesis. However, whether or how nanoFe3O4 affects acetotrophic methanogenesis remain unknown. Herein, we demonstrate the unique role of nanoFe3O4 in accelerating methane production from direct acetotrophic methanogenesis in Methanosarcina-enriched cultrures, which was further confirmed by pure cultures of Methanosarcina barkeri. Compared with other nanomaterials of higher electrical conductivity such as carbon nanotubes (CNTs) and graphite, nanoFe3O4 with mixed valence Fe(II) and Fe(III) had the most significant stimulatory effect on methane production, suggesting its redox activity rather than electrical conductivity led to enhanced methanogenesis by M. barkeri. Cell morphology and spectroscopy analysis revealed that nanoFe3O4 penetrated into the cell membrane and cytoplasm of M. barkeri. These results provide the unprecedented possibility that nanoFe3O4 in the cell membrane of methanogens serve as electron shuttles to facilitate intracellular electron transfer and thus enhance methane production. This work has important implications not only for understanding the mechanisms of mineral-methanogen interaction but also for optimizing engineered methanogenic processes.

Reiner Hedderich - One of the best experts on this subject based on the ideXlab platform.

  • co2 reduction to the level of formylmethanofuran in Methanosarcina barkeri is non energy driven when co is the electron donor
    Fems Microbiology Letters, 2004
    Co-Authors: Alexander Stojanowic, Reiner Hedderich
    Abstract:

    A Methanosarcina barkeri mutant lacking Ech hydrogenase does not catalyze CH4 formation from H2/CO2 since, as was shown previously, the energy-driven reduction of CO2 to formylmethanofuran by H2 is blocked. CH4 formation by this mutant could be restored in the presence of CO or pyruvate. Furthermore, CH4 formation from H2/CO2 plus CO by the Δech mutant was not inhibited by the protonophore TCS. These data show that in vivo the reduction of CO2 to formylmethanofuran can be coupled to the oxidation of CO or pyruvate via a common electron carrier and that the reduction of this electron carrier by H2, catalyzed by Ech hydrogenase, is the energy-driven step in formylmethanofuran-synthesis from CO2, H2 and methanofuran.

  • purification and catalytic properties of ech hydrogenase from Methanosarcina barkeri
    FEBS Journal, 1999
    Co-Authors: Jörn Meuer, Jürgen Koch, Stefan Bartoschek, Andreas Kunkel, Reiner Hedderich
    Abstract:

    Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U·mg protein−1 at pH 7.0 and exhibited an apparent Km for the ferredoxin of 1 µm. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U·mg protein−1 at pH 7.0. The apparent Km for the reduced ferredoxin was 7.5 µm. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent Km for H2 was 5 µm. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H2/CO2-grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO2 during growth on acetate, and to ferredoxin-linked H2 uptake coupled to the reduction of CO2 to the redox state of CO during growth on H2/CO2 or methanol.

  • heterodisulfide reductase from methanol grown cells of Methanosarcina barkeri is not a flavoenzyme
    FEBS Journal, 1997
    Co-Authors: Andreas Kunkel, Rudolf K Thauer, Martin Vaupel, Steffen Heim, Reiner Hedderich
    Abstract:

    Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri (MbHdrDE) is a membrane-bound enzyme composed of a 46-kDa subunit MbHdrD and a 23-kDa subunit MbHdrE. The enzyme has been shown to contain 0.6 mol heme and 20 mol Fe/S per mol heterodimer. In addition, substoichiometric amounts of FAD, thought to be an essential component of the active enzyme, were detected. We have now obtained preparations of active heterodisulfide reductase in high yields completely devoid of a flavin. Cloning and sequencing of the genes encoding MbHdrD and MbHdrE, which were found to form a transcription unit hdrED, revealed that both subunits also lack an FAD-binding motif. MbHdr thus differs from heterodisulifde reductase from Methanobacterium thermoautotrophicum (MtHdr), which is a flavo iron-sulfur protein composed of the subunits MtHdrA (80 kDa), MtHdrB (36 kDa) and MtHdrC (21 kDa), the subunit HdrA harboring the flavin-binding site. Sequence comparisons revealed that the N-terminal third of MbHdrD, which contained two sequence motifs for [4Fe-4S] clusters, is similar to MtHdrC and that the C-terminal two thirds of MbHdrD are similar to MtHdrB. Thus, MbHdrD and MtHdrC are structurally equivalent subunits. MbHdrE shows sequence similarity to b-type cytochromes, in agreement with the finding that this subunit contains a heme. These and other results indicate that MbHdrD harbors the active site of heterodisulfide reduction and that a flavin is not involved in catalysis. Since MbHdrD contains only iron-sulfur clusters, a mechanism of disulfide reduction involving one electron rather than two electron-transfer reactions has to be considered such as operative in ferredoxin :thioredoxin reductases from chloroplasts and cyanobacteria.

  • purification of a two subunit cytochrome b containing heterodisulfide reductase from methanol grown Methanosarcina barkeri
    FEBS Journal, 1994
    Co-Authors: Stefanie Heiden, Reiner Hedderich, Edgar Setzke, Rudolf K Thauer
    Abstract:

    Heterodisulfide reductase catalyzes the terminal step in the energy-conserving electron-transport chain in methanogenic Archaea. The heterodisulfide reductase activity of the membrane fraction of methanol-grown Methanosarcina barkeri was solubilized by Chaps. Chromatography on Q-Sepharose and Superdex-200 yielded a high-molecular-mass fraction (> 700 kDa) which was dissociated by dodecyl β-D-maltoside. After chromatography on Q-Sepharose, an active heterodisulfide reductase preparation was obtained which was composed of only two different subunits of apparent molecular masses 46 kDa and 23 kDa. For each 69 kDa, the enzyme contained 0.6 mol cytochrome b, 0.2 mol FAD, 20 mol non-heme iron and 20 mol acid-labile sulfur. The 23-kDa subunit possessed heme-derived peroxidase activity, showing that this polypeptide is the cytochrome b. The purified enzyme contained the cytochrome b in the reduced form. Upon addition of the heterodisulfide of coenzyme M and N-7-mercaptoheptanoylthreonine phosphate the cytochrome was instantaneously oxidized, indicating that the cytochrome b served as electron donor for heterodisulfide reduction.

Karin Sauer - One of the best experts on this subject based on the ideXlab platform.

  • methanol coenzyme m methyltransferase from Methanosarcina barkeri zinc dependence and thermodynamics of the methanol cob i alamin methyltransferase reaction
    FEBS Journal, 1997
    Co-Authors: Karin Sauer, Rudolf K Thauer
    Abstract:

    In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methyl-coenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated methyltransferases 1 (MT1) and 2 (MT2). Transferase MT1, which is composed of a 50-kDa subunit, MtaB, and a 27-kDa corrinoid-harbouring subunit, MtaC, has been shown recently to catalyze the methylation of free cob(I)alamin with methanol [Sauer, K., Harms, U. & Thauer, R. K. (1997) Eur. J. Biochem. 243, 670-677]. We report here that this reaction is catalyzed by subunit MtaB overproduced in Escherichia coli. MtaB also catalyzed the formation of methanol from methylcobalamin and H2O, the hydrolysis being associated with a free-energy change deltaG(o)' of approximately +7.0 kJ/mol. MtaB was found to contain 1 mol zinc, and its activity to be zinc dependent (pK(Zn2+) = 9.3). The zinc dependence of the MT2 (MtaA)-catalyzed reaction is also described (pK(Zn2+) = 9.6).

  • methanol coenzyme m methyltransferase from Methanosarcina barkeri
    FEBS Journal, 1997
    Co-Authors: Karin Sauer, Ulrike Harms, Rudolf K Thauer
    Abstract:

    The enzyme system catalyzing the formation of methyl-coenzyme M from methanol and coenzyme M in Methanosarcina barkeri is composed of the three different polypeptides MtaA, MtaB and MtaC of which MtaC harbors a corrinoid prosthetic group. The heterologous expression of mtaA and mtaB in Escherichia coli has been described previously. We report here on the overproduction of the apoprotein of MtaC in E. coli, on its reconstitution to the active holoprotein with either cob(II)alamin or methylcob(III)alamin, and on the properties of the reconstituted corrinoid protein. Reconstituted MtaC was found to contain 1 mol bound cobamide/mol. EPR spectroscopic evidence is presented for a His residue as an axial ligand to Co2+of the bound corrinoid. This active-site His was identified by site-directed mutagenesis as His136 in the MtaC sequence that contains four His residues. The reconstituted MtaC, in the cob(I)amide oxidation state, was methylated with methanol in the presence of MtaB and demethylated with coenzyme M in the presence of MtaA. In the presence of both MtaB and MtaA, methyl-coenzyme M was formed from methanol and coenzyme M at specific rates comparable to those determined for the enzyme system purified from M. barkeri. M. barkeri contains an isoenzyme of MtaA designated MtbA. The isoenzyme reacted with MtaC with only 2.5 % of the activity of MtaA.

  • methanol coenzyme m methyltransferase from Methanosarcina barkeri purification properties and encoding genes of the corrinoid protein mt1
    FEBS Journal, 1997
    Co-Authors: Karin Sauer, Ulrike Harms, Rudolf K Thauer
    Abstract:

    The enzyme system catalyzing the formation of methyl-coenzyme M from methanol and coenzyme M in Methanosarcina barkeri is composed of the three different polypeptides MtaA, MtaB and MtaC of which MtaC harbors a corrinoid prosthetic group. The heterologous expression of mtaA and mtaB in Escherichia coli has been described previously. We report here on the overproduction of the apoprotein of MtaC in E. coli, on its reconstitution to the active holoprotein with either cob(II)alamin or methylcob(III)alamin, and on the properties of the reconstituted corrinoid protein. Reconstituted MtaC was found to contain 1 mol bound cobamide/mol. EPR spectroscopic evidence is presented for a His residue as an axial ligand to Co2+of the bound corrinoid. This active-site His was identified by site-directed mutagenesis as His136 in the MtaC sequence that contains four His residues. The reconstituted MtaC, in the cob(I)amide oxidation state, was methylated with methanol in the presence of MtaB and demethylated with coenzyme M in the presence of MtaA. In the presence of both MtaB and MtaA, methyl-coenzyme M was formed from methanol and coenzyme M at specific rates comparable to those determined for the enzyme system purified from M. barkeri. M. barkeri contains an isoenzyme of MtaA designated MtbA. The isoenzyme reacted with MtaC with only 2.5 % of the activity of MtaA.