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Rudolf K Thauer - One of the best experts on this subject based on the ideXlab platform.
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Methyl-CoenzyMe M Reductase froM Methanogenic Archaea: Isotope Effects on Label Exchange and Ethane ForMation with the HoMologous Substrate Ethyl-CoenzyMe M
Journal of the American Chemical Society, 2013Co-Authors: Silvan Scheller, Rudolf K Thauer, Meike Goenrich, Bernhard JaunAbstract:Ethyl-CoenzyMe M (CH3CH2-S-CH2CH2-SO3(-), Et-S-CoM) serves as a hoMologous substrate for the enzyMe Methyl-CoenzyMe M reductase (MCR) resulting in the product ethane instead of Methane. The catalytic reaction proceeds via an interMediate that already contains all six C-H bonds of the product. Because product release occurs after a second, rate-liMiting step, Many cycles of interMediate forMation and reconversion to substrate occur before a substantial aMount of ethane is released. In deuterated buffer, the interMediate becoMes labeled, and C-H activation in the back reaction rapidly leads to labeled Et-S-CoM, which enables interMediate forMation to be detected. Here, we present a coMprehensive analysis of this pre-equilibriuM. (2)H- and (13)C-labeled isotopologues of Et-S-CoM were used as the substrates, and the tiMe course of each isotopologue was followed by NMR spectroscopy. A kinetic siMulation including kinetic isotope effects allowed deterMination of the priMary and α- and β-secondary isotope effects for interMediate forMation and for the C-H/C-D bond activation in the ethane-containing interMediate. The values obtained are in accordance with those found for the native substrate Me-S-CoM (see preceding publication, Scheller, S.; Goenrich, M.; Thauer, R. K.; Jaun, B. J. AM. CheM. Soc. 2013, 135, DOI: 10.1021/ja406485z) and thus iMply the saMe catalytic MechanisM for both substrates. The experiMent by Floss and co-workers, deMonstrating a net inversion of configuration to chiral ethane with CH3CDT-S-CoM as the substrate, is coMpatible with the observed rapid isotope exchange if the isotope effects Measured here are taken into account.
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Methyl-CoenzyMe M reductase froM Methanogenic archaea: isotope effects on the forMation and anaerobic oxidation of Methane.
Journal of the American Chemical Society, 2013Co-Authors: Silvan Scheller, Rudolf K Thauer, Meike Goenrich, Bernhard JaunAbstract:The nickel enzyMe Methyl-CoenzyMe M reductase (MCR) catalyzes two iMportant transforMations in the global carbon cycle: Methane forMation and its reverse, the anaerobic oxidation of Methane. MCR uses the Methyl thioether Methyl-CoenzyMe M (CH3-S-CH2CH2-SO3–, Me-S-CoM) and the thiol CoenzyMe B (CoB-SH) as substrates and converts theM reversibly to Methane and the corresponding heterodisulfide (CoB-S-S-CoM). The catalytic MechanisM is still unknown. Here, we present isotope effects for this reaction in both directions, catalyzed by the enzyMe isolated froM MethanotherMobacter Marburgensis. For Methane forMation, a carbon isotope effect (12CH3-S-CoM/13CH3-S-CoM) of 1.04 ± 0.01 was Measured, showing that breaking of the C–S bond in the substrate Me-S-CoM is the rate-liMiting step. A secondary isotope effect of 1.19 ± 0.01 per D in the Methyl group of CD3-S-CoM indicates a geoMetric change of the Methyl group froM tetrahedral to trigonal planar upon going to the transition state of the rate-liMiting step. This...
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Structure of a Methyl-CoenzyMe M reductase froM Black Sea Mats that oxidize Methane anaerobically
Nature, 2011Co-Authors: Seigo Shima, Rudolf K Thauer, Jörg Kahnt, Martin Krueger, Tobias Weinert, Ulrike Demmer, Ulrich ErmlerAbstract:The anaerobic oxidation of Methane (AOM) with sulphate, an area currently generating great interest in Microbiology, is accoMplished by consortia of Methanotrophic archaea (ANME) and sulphate-reducing bacteria1, 2. The enzyMe activating Methane in Methanotrophic archaea has tentatively been identified as a hoMologue of Methyl-CoenzyMe M reductase (MCR) that catalyses the Methane-forMing step in Methanogenic archaea3, 4. Here we report an X-ray structure of the 280 kDa heterohexaMeric ANME-1 MCR coMplex. It was crystallized uniquely froM a protein enseMble purified froM consortia of MicroorganisMs collected with a subMersible froM a Black Sea Mat catalysing AOM with sulphate4. Crystals grown froM the heterogeneous saMple diffract to 2.1 A resolution and consist of a single ANME-1 MCR population, deMonstrating the strong selective power of crystallization. The structure revealed ANME-1 MCR in coMplex with CoenzyMe M and CoenzyMe B, indicating the saMe substrates for MCR froM Methanotrophic and Methanogenic archaea. Differences between the highly siMilar structures of ANME-1 MCR and Methanogenic MCR include a F430 Modification, a cysteine-rich patch and an altered post-translational aMino acid Modification pattern, which May tune the enzyMes for their functions in different biological contexts.
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Binding of CoenzyMe B induces a Major conforMational change in the active site of Methyl-CoenzyMe M reductase.
Journal of the American Chemical Society, 2010Co-Authors: Sieglinde Ebner, Bernhard Jaun, Meike Goenrich, Rudolf K Thauer, Jeffrey HarmerAbstract:Methyl-CoenzyMe M reductase (MCR) is the key enzyMe in Methane forMation by Methanogenic Archaea. It converts the thioether Methyl-CoenzyMe M and the thiol CoenzyMe B into Methane and the heterodisulfide of CoenzyMe M and CoenzyMe B. The catalytic MechanisM of MCR and the role of its prosthetic group, the nickel hydrocorphin CoenzyMe F430, is still disputed, and no interMediates have been observed so far by fast spectroscopic techniques when the enzyMe was incubated with the natural substrates. In the presence of the coMpetitive inhibitor CoenzyMe M instead of Methyl-CoenzyMe M, addition of CoenzyMe B to the active Ni(I) state MCRred1 induces two new species called MCRred2a and MCRred2r which have been characterized by pulse EPR spectroscopy. Here we show that the two MCRred2 signals can also be induced by the S-Methyl- and the S-trifluoroMethyl analogs of CoenzyMe B. 19F-ENDOR data for MCRred2a and MCRred2r induced by S-CF3-CoenzyMe B show that, upon binding of the CoenzyMe B analog, the end of the 7-thi...
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Nickel-alkyl bond forMation in the active site of Methyl-CoenzyMe M reductase.
Metal ions in life sciences, 2009Co-Authors: Bernhard Jaun, Rudolf K ThauerAbstract:Methyl-CoenzyMe M reductase (MCR) catalyzes the Methane-forMing step in Methanogenic archaea and Most probably also the Methane-oxidizing step in Methanotrophic archaea. The enzyMe contains CoenzyMe F430 as prosthetic group. F430 is a nickel porphinoid that has to be in the reduced Ni(I) state for the enzyMe to be active. The presently discussed catalytic MechanisMs of MCR can in principle be divided into two basic Models. In one Model the key interMediate features a Methyl-Ni(III) species being either forMed in a nucleophilic substitution reaction or in an oxidative addition reaction. In the other Model first the thioether sulfur of Methyl-CoenzyMe M binds to the Ni(I), which subsequently results in the release of the Methyl group as Methyl radical leaving behind a Ni(II)-sulfur bond. The experiMental evidence for and against a Methyl-nickel interMediate is reviewed.
Evert C Duin - One of the best experts on this subject based on the ideXlab platform.
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asseMbly of Methyl CoenzyMe M reductase in the Methanogenic archaeon Methanococcus Maripaludis
Journal of Bacteriology, 2018Co-Authors: Chauwen Chou, Robel Ghebreab, Evert C Duin, Liangliang Wang, Dennis Phillips, William B. WhitmanAbstract:ABSTRACT Methyl CoenzyMe M reductase (MCR) is a coMplex enzyMe that catalyzes the final step in biological Methanogenesis. To better understand its asseMbly, the recoMbinant MCR froM the therMophile MethanotherMococcus okinawensis (rMCR ok ) was expressed in the Mesophile Methanococcus Maripaludis. The rMCR ok was posttranslationally Modified correctly and contained McrD and the unique nickel tetrapyrrole CoenzyMe F 430 . Subunits of the native M. Maripaludis (MCR Mar ) were largely absent, suggesting that the recoMbinant enzyMe was forMed by an asseMbly of cotranscribed subunits. Strong support for this hypothesis was obtained by expressing a chiMeric operon coMprising the His-tagged McrA froM M. Maripaludis and the McrBDCG froM M. okinawensis in M. Maripaludis. The His-tagged purified rMCR then contained the M. Maripaludis McrA and the M. okinawensis McrBDG. The present study proMpted us to forM a working Model for MCR asseMbly, which can be further tested by the heterologous expression systeM established here. IMPORTANCE ApproxiMately 1.6% of the net priMary production of plants, algae, and cyanobacteria are processed by biological Methane production in anoxic environMents. This accounts for about 74% of the total global Methane production, up to 25% of which is consuMed by anaerobic oxidation of Methane (AOM). Methyl CoenzyMe M reductase (MCR) is the key enzyMe in both Methanogenesis and AOM. MCR is asseMbled as a diMer of two heterotriMers, where posttranslational Modifications and F 430 cofactors are eMbedded in the active sites. However, this coMplex asseMbly process reMains unknown. Here, we established a heterologous expression systeM for MCR to learn how MCR is asseMbled.
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Elucidating the Process of Activation of Methyl-CoenzyMe M Reductase
Journal of Bacteriology, 2014Co-Authors: Divya Prakash, Yonnie Wu, Evert C DuinAbstract:Methyl-CoenzyMe M reductase (MCR) catalyzes the reversible reduction of Methyl-CoenzyMe M (CH3-S-CoM) and CoenzyMe B (HS-CoB) to Methane and heterodisulfide CoM-S-S-CoB (HDS). MCR contains the hydroporphinoid nickel coMplex CoenzyMe F430 in its active site, and the Ni center has to be in its Ni(I) valence state for the enzyMe to be active. Until now, no in vitro Method that fully converted the inactive MCRsilent-Ni(II) forM to the active MCRred1-Ni(I) forM has been described. With the potential use of recoMbinant MCR in the production of biofuels and the need to better understand this enzyMe and its activation process, we studied its activation under nonturnover conditions and achieved full MCR activation in the presence of dithiothreitol and protein coMponents A2, an ATP carrier, and A3a. It was found that the presence of HDS proMotes the inactivation of MCRred1, which Makes it essential that the activation process is isolated froM the Methane forMation assay, which tends to result in MiniMal activation rates. CoMponent A3a is a MultienzyMe coMplex that includes the McrC gene product, an Fe-protein hoMolog, an iron-sulfur flavoprotein, and protein coMponents involved in electron bifurcation. A hypothetical Model for the cellular activation process of MCR is presented.
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Methyl-CoenzyMe M reductase froM MethanotherMobacter Marburgensis.
Methods in enzymology, 2011Co-Authors: Evert C Duin, Divya Prakash, Charlene BrungessAbstract:Methyl-CoenzyMe M reductase catalyzes the reversible synthesis of Methane froM Methyl-CoenzyMe M in Methanogenic and ANME-1 and ANME-2 Archaea. The purification procedure for Methyl-CoenzyMe M reductase froM MethanotherMobacter Marburgensis is described. The procedure is an accuMulation of alMost 30 years of research on MCR starting with the first purification described by Ellefson and Wolfe (Ellefson, W.L., and Wolfe, R.S. (1981). CoMponent C of the Methylreductase systeM of MethanobacteriuM. J. Biol. CheM.256, 4259-4262). To provide a context for this procedure, soMe background inforMation is provided, including a description of whole cell experiMents that provided Much of our knowledge of the behavior and properties of Methyl-CoenzyMe M reductase.
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Handbook of Metalloproteins - Methyl‐CoenzyMe M Reductase
Handbook of Metalloproteins, 2006Co-Authors: Wolfgang Grabarse, Evert C Duin, Seigo Shima, Rudolf K Thauer, Felix Mahlert, Ulrich ErmlerAbstract:Functional Class Occurrence Biological Function AMino Acid Sequence InforMation Metal Content and Cofactors Protein Production, Purification, Molecular Characterization Activity Test Spectroscopy X-Ray Structure of Inactive MCR Functional Aspects Functional Derivatives Possible Catalytic MechanisM AcknowledgeMents Related Articles 3D Structure Keywords: Methanogenesis; nickel; tetrapyrrole; CoenzyMe M; CoenzyMe B; thioether cleavage; radical interMediates; Modified aMino acids
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Spin density and CoenzyMe M coordination geoMetry of the ox1 forM of Methyl-CoenzyMe M reductase: a pulse EPR study.
Journal of the American Chemical Society, 2005Co-Authors: Jeffrey Harmer, Bernhard Jaun, Evert C Duin, Meike Goenrich, Rudolf K Thauer, Cinzia Finazzo, Carsten Bauer, Rafal Piskorski, Sabine Van Doorslaer, Arthur SchweigerAbstract:Methyl-CoenzyMe M reductase (MCR) catalyses the reduction of Methyl-CoenzyMe M (CH3-S-CoM) with CoenzyMe B (H-S-CoB) to CH4 and CoM-S-S-CoB in Methanogenic archaea. Here we present a pulse EPR stud...
Bernhard Jaun - One of the best experts on this subject based on the ideXlab platform.
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Methyl-CoenzyMe M reductase froM Methanogenic archaea: isotope effects on the forMation and anaerobic oxidation of Methane.
Journal of the American Chemical Society, 2013Co-Authors: Silvan Scheller, Rudolf K Thauer, Meike Goenrich, Bernhard JaunAbstract:The nickel enzyMe Methyl-CoenzyMe M reductase (MCR) catalyzes two iMportant transforMations in the global carbon cycle: Methane forMation and its reverse, the anaerobic oxidation of Methane. MCR uses the Methyl thioether Methyl-CoenzyMe M (CH3-S-CH2CH2-SO3–, Me-S-CoM) and the thiol CoenzyMe B (CoB-SH) as substrates and converts theM reversibly to Methane and the corresponding heterodisulfide (CoB-S-S-CoM). The catalytic MechanisM is still unknown. Here, we present isotope effects for this reaction in both directions, catalyzed by the enzyMe isolated froM MethanotherMobacter Marburgensis. For Methane forMation, a carbon isotope effect (12CH3-S-CoM/13CH3-S-CoM) of 1.04 ± 0.01 was Measured, showing that breaking of the C–S bond in the substrate Me-S-CoM is the rate-liMiting step. A secondary isotope effect of 1.19 ± 0.01 per D in the Methyl group of CD3-S-CoM indicates a geoMetric change of the Methyl group froM tetrahedral to trigonal planar upon going to the transition state of the rate-liMiting step. This...
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Methyl-CoenzyMe M Reductase froM Methanogenic Archaea: Isotope Effects on Label Exchange and Ethane ForMation with the HoMologous Substrate Ethyl-CoenzyMe M
Journal of the American Chemical Society, 2013Co-Authors: Silvan Scheller, Rudolf K Thauer, Meike Goenrich, Bernhard JaunAbstract:Ethyl-CoenzyMe M (CH3CH2-S-CH2CH2-SO3(-), Et-S-CoM) serves as a hoMologous substrate for the enzyMe Methyl-CoenzyMe M reductase (MCR) resulting in the product ethane instead of Methane. The catalytic reaction proceeds via an interMediate that already contains all six C-H bonds of the product. Because product release occurs after a second, rate-liMiting step, Many cycles of interMediate forMation and reconversion to substrate occur before a substantial aMount of ethane is released. In deuterated buffer, the interMediate becoMes labeled, and C-H activation in the back reaction rapidly leads to labeled Et-S-CoM, which enables interMediate forMation to be detected. Here, we present a coMprehensive analysis of this pre-equilibriuM. (2)H- and (13)C-labeled isotopologues of Et-S-CoM were used as the substrates, and the tiMe course of each isotopologue was followed by NMR spectroscopy. A kinetic siMulation including kinetic isotope effects allowed deterMination of the priMary and α- and β-secondary isotope effects for interMediate forMation and for the C-H/C-D bond activation in the ethane-containing interMediate. The values obtained are in accordance with those found for the native substrate Me-S-CoM (see preceding publication, Scheller, S.; Goenrich, M.; Thauer, R. K.; Jaun, B. J. AM. CheM. Soc. 2013, 135, DOI: 10.1021/ja406485z) and thus iMply the saMe catalytic MechanisM for both substrates. The experiMent by Floss and co-workers, deMonstrating a net inversion of configuration to chiral ethane with CH3CDT-S-CoM as the substrate, is coMpatible with the observed rapid isotope exchange if the isotope effects Measured here are taken into account.
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Binding of CoenzyMe B induces a Major conforMational change in the active site of Methyl-CoenzyMe M reductase.
Journal of the American Chemical Society, 2010Co-Authors: Sieglinde Ebner, Bernhard Jaun, Meike Goenrich, Rudolf K Thauer, Jeffrey HarmerAbstract:Methyl-CoenzyMe M reductase (MCR) is the key enzyMe in Methane forMation by Methanogenic Archaea. It converts the thioether Methyl-CoenzyMe M and the thiol CoenzyMe B into Methane and the heterodisulfide of CoenzyMe M and CoenzyMe B. The catalytic MechanisM of MCR and the role of its prosthetic group, the nickel hydrocorphin CoenzyMe F430, is still disputed, and no interMediates have been observed so far by fast spectroscopic techniques when the enzyMe was incubated with the natural substrates. In the presence of the coMpetitive inhibitor CoenzyMe M instead of Methyl-CoenzyMe M, addition of CoenzyMe B to the active Ni(I) state MCRred1 induces two new species called MCRred2a and MCRred2r which have been characterized by pulse EPR spectroscopy. Here we show that the two MCRred2 signals can also be induced by the S-Methyl- and the S-trifluoroMethyl analogs of CoenzyMe B. 19F-ENDOR data for MCRred2a and MCRred2r induced by S-CF3-CoenzyMe B show that, upon binding of the CoenzyMe B analog, the end of the 7-thi...
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Nickel-alkyl bond forMation in the active site of Methyl-CoenzyMe M reductase.
Metal ions in life sciences, 2009Co-Authors: Bernhard Jaun, Rudolf K ThauerAbstract:Methyl-CoenzyMe M reductase (MCR) catalyzes the Methane-forMing step in Methanogenic archaea and Most probably also the Methane-oxidizing step in Methanotrophic archaea. The enzyMe contains CoenzyMe F430 as prosthetic group. F430 is a nickel porphinoid that has to be in the reduced Ni(I) state for the enzyMe to be active. The presently discussed catalytic MechanisMs of MCR can in principle be divided into two basic Models. In one Model the key interMediate features a Methyl-Ni(III) species being either forMed in a nucleophilic substitution reaction or in an oxidative addition reaction. In the other Model first the thioether sulfur of Methyl-CoenzyMe M binds to the Ni(I), which subsequently results in the release of the Methyl group as Methyl radical leaving behind a Ni(II)-sulfur bond. The experiMental evidence for and against a Methyl-nickel interMediate is reviewed.
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Coordination and binding geoMetry of Methyl-CoenzyMe M in the red1M state of Methyl-CoenzyMe M reductase
Journal of Biological Inorganic Chemistry, 2008Co-Authors: Dariush Hinderberger, Bernhard Jaun, Stefan Mayr, Sieglinde Ebner, Meike Goenrich, Markus Reiher, Rudolf K Thauer, Jeffrey HarmerAbstract:Methane forMation in Methanogenic Archaea is catalyzed by Methyl-CoenzyMe M reductase (MCR) and takes place via the reduction of Methyl-CoenzyMe M (CH3-S-CoM) with CoenzyMe B (HS-CoB) to Methane and the heterodisulfide CoM-S-S-CoB. MCR harbors the nickel porphyrinoid CoenzyMe F430 as a prosthetic group, which has to be in the Ni(I) oxidation state for the enzyMe to be active. To date no interMediates in the catalytic cycle of MCRred1 (red for reduced Ni) have been identified. Here, we report a detailed characterization of MCRred1M (''M'' for Methyl-CoenzyMe M), which is the coMplex of MCRred1a (''a'' for absence of substrate) with CH3-S-CoM. Using continuous-wave and pulse electron paraMagnetic reso- nance spectroscopy in coMbination with selective isotope labeling ( 13 C and 2 H) of CH3-S-CoM, it is shown that CH3-S-CoM binds in the active site of MCR such that its thioether sulfur is weakly coordinated to the Ni(I) of F430. The coMplex is stable until the addition of the second sub- strate, HS-CoB. Results froM EPR spectroscopy, along with quantuM Mechanical calculations, are used to characterize the electronic and geoMetric structure of this coMplex, which can be regarded as the first interMediate in the catalytic MechanisM.
Meike Goenrich - One of the best experts on this subject based on the ideXlab platform.
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Methyl-CoenzyMe M reductase froM Methanogenic archaea: isotope effects on the forMation and anaerobic oxidation of Methane.
Journal of the American Chemical Society, 2013Co-Authors: Silvan Scheller, Rudolf K Thauer, Meike Goenrich, Bernhard JaunAbstract:The nickel enzyMe Methyl-CoenzyMe M reductase (MCR) catalyzes two iMportant transforMations in the global carbon cycle: Methane forMation and its reverse, the anaerobic oxidation of Methane. MCR uses the Methyl thioether Methyl-CoenzyMe M (CH3-S-CH2CH2-SO3–, Me-S-CoM) and the thiol CoenzyMe B (CoB-SH) as substrates and converts theM reversibly to Methane and the corresponding heterodisulfide (CoB-S-S-CoM). The catalytic MechanisM is still unknown. Here, we present isotope effects for this reaction in both directions, catalyzed by the enzyMe isolated froM MethanotherMobacter Marburgensis. For Methane forMation, a carbon isotope effect (12CH3-S-CoM/13CH3-S-CoM) of 1.04 ± 0.01 was Measured, showing that breaking of the C–S bond in the substrate Me-S-CoM is the rate-liMiting step. A secondary isotope effect of 1.19 ± 0.01 per D in the Methyl group of CD3-S-CoM indicates a geoMetric change of the Methyl group froM tetrahedral to trigonal planar upon going to the transition state of the rate-liMiting step. This...
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Methyl-CoenzyMe M Reductase froM Methanogenic Archaea: Isotope Effects on Label Exchange and Ethane ForMation with the HoMologous Substrate Ethyl-CoenzyMe M
Journal of the American Chemical Society, 2013Co-Authors: Silvan Scheller, Rudolf K Thauer, Meike Goenrich, Bernhard JaunAbstract:Ethyl-CoenzyMe M (CH3CH2-S-CH2CH2-SO3(-), Et-S-CoM) serves as a hoMologous substrate for the enzyMe Methyl-CoenzyMe M reductase (MCR) resulting in the product ethane instead of Methane. The catalytic reaction proceeds via an interMediate that already contains all six C-H bonds of the product. Because product release occurs after a second, rate-liMiting step, Many cycles of interMediate forMation and reconversion to substrate occur before a substantial aMount of ethane is released. In deuterated buffer, the interMediate becoMes labeled, and C-H activation in the back reaction rapidly leads to labeled Et-S-CoM, which enables interMediate forMation to be detected. Here, we present a coMprehensive analysis of this pre-equilibriuM. (2)H- and (13)C-labeled isotopologues of Et-S-CoM were used as the substrates, and the tiMe course of each isotopologue was followed by NMR spectroscopy. A kinetic siMulation including kinetic isotope effects allowed deterMination of the priMary and α- and β-secondary isotope effects for interMediate forMation and for the C-H/C-D bond activation in the ethane-containing interMediate. The values obtained are in accordance with those found for the native substrate Me-S-CoM (see preceding publication, Scheller, S.; Goenrich, M.; Thauer, R. K.; Jaun, B. J. AM. CheM. Soc. 2013, 135, DOI: 10.1021/ja406485z) and thus iMply the saMe catalytic MechanisM for both substrates. The experiMent by Floss and co-workers, deMonstrating a net inversion of configuration to chiral ethane with CH3CDT-S-CoM as the substrate, is coMpatible with the observed rapid isotope exchange if the isotope effects Measured here are taken into account.
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Binding of CoenzyMe B induces a Major conforMational change in the active site of Methyl-CoenzyMe M reductase.
Journal of the American Chemical Society, 2010Co-Authors: Sieglinde Ebner, Bernhard Jaun, Meike Goenrich, Rudolf K Thauer, Jeffrey HarmerAbstract:Methyl-CoenzyMe M reductase (MCR) is the key enzyMe in Methane forMation by Methanogenic Archaea. It converts the thioether Methyl-CoenzyMe M and the thiol CoenzyMe B into Methane and the heterodisulfide of CoenzyMe M and CoenzyMe B. The catalytic MechanisM of MCR and the role of its prosthetic group, the nickel hydrocorphin CoenzyMe F430, is still disputed, and no interMediates have been observed so far by fast spectroscopic techniques when the enzyMe was incubated with the natural substrates. In the presence of the coMpetitive inhibitor CoenzyMe M instead of Methyl-CoenzyMe M, addition of CoenzyMe B to the active Ni(I) state MCRred1 induces two new species called MCRred2a and MCRred2r which have been characterized by pulse EPR spectroscopy. Here we show that the two MCRred2 signals can also be induced by the S-Methyl- and the S-trifluoroMethyl analogs of CoenzyMe B. 19F-ENDOR data for MCRred2a and MCRred2r induced by S-CF3-CoenzyMe B show that, upon binding of the CoenzyMe B analog, the end of the 7-thi...
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Coordination and binding geoMetry of Methyl-CoenzyMe M in the red1M state of Methyl-CoenzyMe M reductase
Journal of Biological Inorganic Chemistry, 2008Co-Authors: Dariush Hinderberger, Bernhard Jaun, Stefan Mayr, Sieglinde Ebner, Meike Goenrich, Markus Reiher, Rudolf K Thauer, Jeffrey HarmerAbstract:Methane forMation in Methanogenic Archaea is catalyzed by Methyl-CoenzyMe M reductase (MCR) and takes place via the reduction of Methyl-CoenzyMe M (CH3-S-CoM) with CoenzyMe B (HS-CoB) to Methane and the heterodisulfide CoM-S-S-CoB. MCR harbors the nickel porphyrinoid CoenzyMe F430 as a prosthetic group, which has to be in the Ni(I) oxidation state for the enzyMe to be active. To date no interMediates in the catalytic cycle of MCRred1 (red for reduced Ni) have been identified. Here, we report a detailed characterization of MCRred1M (''M'' for Methyl-CoenzyMe M), which is the coMplex of MCRred1a (''a'' for absence of substrate) with CH3-S-CoM. Using continuous-wave and pulse electron paraMagnetic reso- nance spectroscopy in coMbination with selective isotope labeling ( 13 C and 2 H) of CH3-S-CoM, it is shown that CH3-S-CoM binds in the active site of MCR such that its thioether sulfur is weakly coordinated to the Ni(I) of F430. The coMplex is stable until the addition of the second sub- strate, HS-CoB. Results froM EPR spectroscopy, along with quantuM Mechanical calculations, are used to characterize the electronic and geoMetric structure of this coMplex, which can be regarded as the first interMediate in the catalytic MechanisM.
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Two sub-states of the red2 state of Methyl-CoenzyMe M reductase revealed by high-field EPR spectroscopy
JBIC Journal of Biological Inorganic Chemistry, 2007Co-Authors: Denise I. Kern, Bernhard Jaun, Meike Goenrich, Jeffrey Harmer, Rudolf K Thauer, Dariush HinderbergerAbstract:Methyl-CoenzyMe M reductase (MCR) catalyzes the forMation of Methane froM Methyl-CoenzyMe M and CoenzyMe B in Methanogenic archaea. The enzyMe has two structurally interlinked active sites eMbedded in an α_2β_2γ_2 subunit structure. Each active site has the nickel porphyrinoid F_430 as a prosthetic group. In the active state, F_430 contains the transition Metal in the Ni(I) oxidation state. The active enzyMe exhibits an axial Ni(I)-based continuous wave (CW) electron paraMagnetic resonance (EPR) signal, called red1a in the absence of substrates or red1c in the presence of CoenzyMe M. Addition of CoenzyMe B to the MCR-red1 state can partially and reversibly convert it into the MCR-red2 forM, which shows a rhoMbic Ni(I)-based EPR signal (at X-band Microwave frequencies of approxiMately 9.4 GHz). In this report we present evidence froM high-field/high-frequency CW EPR spectroscopy (W-band, Microwave frequency of approxiMately 94 GHz) that the red2 state consists of two substates that could not be resolved by EPR spectroscopy at X-band frequencies. At W-band it becoMes apparent that upon addition of CoenzyMe B to MCR in the red1c state, two red2 EPR signals are induced, not one as was previously believed. The first signal is the well-characterized (ortho)rhoMbic EPR signal, thus far called red2, while the second previously unidentified signal is axial. We have naMed the two substates MCR-red2r and MCR-red2a after their rhoMbic and axial signals, respectively.
Stephen W Ragsdale - One of the best experts on this subject based on the ideXlab platform.
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In vivo activation of Methyl-CoenzyMe M reductase by carbon Monoxide.
Frontiers in Microbiology, 2013Co-Authors: Yuzhen Zhou, Alexandria E. Dorchak, Stephen W RagsdaleAbstract:Methyl-CoenzyMe M reductase (MCR) froM Methanogenic archaea catalyzes the rate-liMiting and final step in Methane biosynthesis. Using CoenzyMe B (CoBSH) as the two-electron donor, MCR reduces Methyl-CoenzyMe M (CH3-SCoM) to Methane and the Mixed disulfide, CoBS-SCoM. MCR contains an essential redox-active nickel tetrahydro¬corphinoid cofactor, CoenzyMe F430, at its active site. The active forM of the enzyMe (MCRred1) contains Ni(I)-F430. Rapid and efficient conversion of MCR to MCRred1 is iMportant for elucidating the enzyMatic MechanisM, yet this reduction is difficult because the Ni(I) state is subject to oxidative inactivation. FurtherMore, no in vitro Methods have yet been described to convert Ni(II) forMs into MCRred1. Since 1991, it has been known that MCRred1 froM MethanotherMobacter Marburgensis can be generated in vivo when cells are purged with 100% H2. Here we show that purging cells or cell extracts with CO can also activate MCR. The rate of in vivo activation by CO is about 15 tiMes faster than by H2 (130 Min-1 and 8 Min-1, respectively) and CO leads to two-fold higher MCRred1 than H2. Unlike H2-dependent activation, which exhibits a 10-h lag tiMe, there is no lag for CO-dependent activation. Based on cyanide inhibition experiMents, CODH is required for the CO-dependent activation. ForMate, which also is a strong reductant, cannot activate MCR in M. Marburgensis in vivo.
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Detection of organoMetallic and radical interMediates in the catalytic MechanisM of Methyl-CoenzyMe M reductase using the natural substrate Methyl-CoenzyMe M and a CoenzyMe B substrate analogue.
Biochemistry, 2010Co-Authors: Xianghui Li, Ryan C Kunz, Stephen W RagsdaleAbstract:Methyl-CoenzyMe M reductase (MCR) froM Methanogenic archaea catalyzes the terMinal step in Methanogenesis using CoenzyMe B (CoBSH) as the two-electron donor to reduce Methyl-CoenzyMe M (Methyl-SCoM) to forM Methane and the heterodisulfide, CoBS-SCoM. The active site of MCR contains an essential redox-active nickel tetrapyrrole cofactor, CoenzyMe F430, which is active in the Ni(I) state (MCRred1). Several catalytic MechanisMs have been proposed for Methane synthesis that Mainly differ in whether an organoMetallic Methyl-Ni(III) or a Methyl radical is the first catalytic interMediate. A MechanisM was recently proposed in which Methyl-Ni(III) undergoes hoMolysis to generate a Methyl radical (Li, X., Telser, J., Kunz, R. C., HoffMan, B. M., Gerfen, G., and Ragsdale, S. W. (2010) BiocheMistry 49, 6866−6876). DiscriMination aMong these MechanisMs requires identification of the proposed interMediates, none of which have been observed with native substrates. Apparently, interMediates forM and decay too rapidly to...
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Structural Insight into Methyl-CoenzyMe M Reductase CheMistry Using CoenzyMe B Analogues,
Biochemistry, 2010Co-Authors: Peder E. Cedervall, Arwen R. Pearson, Stephen W Ragsdale, Carrie M. WilmotAbstract:Methyl-CoenzyMe M reductase (MCR) catalyzes the final and rate-liMiting step in Methane biogenesis: the reduction of Methyl-CoenzyMe M (Methyl-SCoM) by CoenzyMe B (CoBSH) to Methane and a heterodisulfide (CoBS-SCoM). Crystallographic studies show that the active site is deeply buried within the enzyMe and contains a highly reduced nickel-tetrapyrrole, CoenzyMe F430. Methyl-SCoM Must enter the active site prior to CoBSH, as species derived froM Methyl-SCoM are always observed bound to the F430 nickel in the deepest part of the 30 A long substrate channel that leads froM the protein surface to the active site. The seven-carbon Mercaptoalkanoyl chain of CoBSH binds within a 16 A predoMinantly hydrophobic part of the channel close to F430, with the CoBSH thiolate lying closest to the nickel at a distance of 8.8 A. It has previously been suggested that binding of CoBSH initiates catalysis by inducing a conforMational change that Moves Methyl-SCoM closer to the nickel proMoting cleavage of the C−S bond of Methy...
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Observation of organoMetallic and radical interMediates forMed during the reaction of Methyl-CoenzyMe M reductase with broMoethanesulfonate
Biochemistry, 2010Co-Authors: Joshua Telser, Ryan C Kunz, Brian M. Hoffman, Gary J. Gerfen, Stephen W RagsdaleAbstract:Methyl-CoenzyMe M reductase (MCR) froM Methanogenic archaea catalyzes the final step of Methane forMation, in which Methyl-CoenzyMe M (2-Methylthioethanesulfonate, Methyl-SCoM) is reduced with coen...
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GeoMetric and electronic structures of the Ni(I) and Methyl-Ni(III) interMediates of Methyl-CoenzyMe M reductase.
Biochemistry, 2009Co-Authors: Ritimukta Sarangi, Mishtu Dey, Stephen W RagsdaleAbstract:Methyl-CoenzyMe M reductase (MCR) catalyzes the terMinal step in the forMation of biological Methane froM Methyl-CoenzyMe M (Me-SCoM) and CoenzyMe B (CoBSH). The active site in MCR contains a Ni−F430 cofactor, which can exist in different oxidation states. The catalytic MechanisM of Methane forMation has reMained elusive despite intense spectroscopic and theoretical investigations. On the basis of spectroscopic and crystallographic data, the first step of the MechanisM is proposed to involve a nucleophilic attack of the NiI active state (MCRred1) on Me-SCoM to forM a NiIII−Methyl interMediate, while coMputational studies indicate that the first step involves the attack of NiI on the sulfur of Me-SCoM, forMing a CH3• radical and a NiII−thiolate species. In this study, a coMbination of Ni K-edge X-ray absorption spectroscopic (XAS) studies and density functional theory (DFT) calculations have been perforMed on the NiI (MCRred1), NiII (MCRred1−silent), and NiIII−Methyl (MCRMe) states of MCR to elucidate the ...