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Gabriele Diekert - One of the best experts on this subject based on the ideXlab platform.
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Redox potential changes during ATP‐dependent Corrinoid reduction determined by redox titrations with europium(II)–DTPA
Protein science : a publication of the Protein Society, 2019Co-Authors: Hendrike Dürichen, Gabriele Diekert, Sandra StudenikAbstract:Corrinoids are essential cofactors of enzymes involved in the C1 metabolism of anaerobes. The active, super-reduced [CoI ] state of the Corrinoid cofactor is highly sensitive to autoxidation. In O-demethylases, the oxidation to inactive [CoII ] is reversed by an ATP-dependent electron transfer catalyzed by the activating enzyme (AE). The redox potential changes of the Corrinoid cofactor, which occur during this reaction, were studied by potentiometric titration coupled to UV/visible spectroscopy. By applying europium(II)-diethylenetriaminepentaacetic acid (DTPA) as a reductant, we were able to determine the midpoint potential of the [CoII ]/[CoI ] couple of the protein-bound Corrinoid cofactor in the absence and presence of AE and/or ATP. The data revealed that the transfer of electrons from a physiological donor to the Corrinoid as the electron-accepting site is achieved by increasing the potential of the Corrinoid cofactor from -530 ± 15 mV to -250 ± 10 mV (ESHE , pH 7.5). The first 50 to 100 mV of the shift of the redox potential seem to be caused by the interaction of nucleotide-bound AE with the Corrinoid protein or its cofactor. The remaining 150-200 mV had to be overcome by the chemical energy of ATP hydrolysis. The experiments revealed that Eu(II)-DTPA, which was already known as a powerful reducing agent, is a suitable electron donor for titration experiments of low-potential redox centers. Furthermore, the results of this study will contribute to the understanding of thermodynamically unfavorable electron transfer processes driven by the power of ATP hydrolysis.
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redox potential changes during atp dependent Corrinoid reduction determined by redox titrations with europium ii dtpa
Protein Science, 2019Co-Authors: Hendrike Dürichen, Gabriele Diekert, Sandra StudenikAbstract:Corrinoids are essential cofactors of enzymes involved in the C1 metabolism of anaerobes. The active, super-reduced [CoI ] state of the Corrinoid cofactor is highly sensitive to autoxidation. In O-demethylases, the oxidation to inactive [CoII ] is reversed by an ATP-dependent electron transfer catalyzed by the activating enzyme (AE). The redox potential changes of the Corrinoid cofactor, which occur during this reaction, were studied by potentiometric titration coupled to UV/visible spectroscopy. By applying europium(II)-diethylenetriaminepentaacetic acid (DTPA) as a reductant, we were able to determine the midpoint potential of the [CoII ]/[CoI ] couple of the protein-bound Corrinoid cofactor in the absence and presence of AE and/or ATP. The data revealed that the transfer of electrons from a physiological donor to the Corrinoid as the electron-accepting site is achieved by increasing the potential of the Corrinoid cofactor from -530 ± 15 mV to -250 ± 10 mV (ESHE , pH 7.5). The first 50 to 100 mV of the shift of the redox potential seem to be caused by the interaction of nucleotide-bound AE with the Corrinoid protein or its cofactor. The remaining 150-200 mV had to be overcome by the chemical energy of ATP hydrolysis. The experiments revealed that Eu(II)-DTPA, which was already known as a powerful reducing agent, is a suitable electron donor for titration experiments of low-potential redox centers. Furthermore, the results of this study will contribute to the understanding of thermodynamically unfavorable electron transfer processes driven by the power of ATP hydrolysis.
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exogenous 5 6 dimethylbenzimidazole caused production of a non functional tetrachloroethene reductive dehalogenase in sulfurospirillum multivorans
Environmental Microbiology, 2014Co-Authors: Sebastian Keller, Bernhard Krautler, Gabriele Diekert, Markus Ruetz, Cindy Kunze, Torsten SchubertAbstract:Summary Corrinoid-dependent reductive dehalogenation is mediated by phylogenetically diverse anaerobic bacteria that either synthesize Corrinoids de novo or are dependent on Corrinoid salvaging from the environment. The tetrachloroethene (PCE) reduc- tive dehalogenase (PceA) of the Gram-negative Epsilonproteobacterium Sulfurospirillum multivorans harbours a norpseudo-B12 as Corrinoid cofactor. Norpseudo-B12 differs from coenzyme B12 in the nucleotide loop structure. Adenine instead of 5,6- dimethylbenzimidazole (DMB) serves as lower ligand base of the central cobalt ion, and the nucleotide loop of norpseudo-B12 lacks a methyl group at position 176. In this study, S. multivorans was grown anaerobically with PCE in the presence of DMB. At a DMB concentration of 25 M, the adenine moiety in the nucleotide loop of norpseudo-B12 was quantita- tively replaced by DMB. The formation of the DMB- containing nor-B12 severely affected PCE-dependent growth and the PceA activity. In DMB-treated cells processing of the cytoplasmic PceA precursor was impeded, a result pointing to retarded cofactor incorporation. PceA enriched from cells cultivated with DMB contained nor-B12. Nor-B12 purified from cells grown in the presence of DMB mediated the abiotic reductive dehalogenation of trichloroacetate to dichloroacetate at a 25-fold lower rate in compari-
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kinetic regulation of a Corrinoid reducing metallo atpase by its substrates
Molecular Microbiology, 2014Co-Authors: Martin Sperfeld, Gabriele Diekert, Sandra StudenikAbstract:Summary Corrinoid cofactors play a crucial role as methyl group carriers in the C1 metabolism of anaerobes, e.g. in the cleavage of phenyl methyl ethers by O-demethylases. For the methylation, the protein-bound Corrinoid has to be in the super-reduced [CoI]-state, which is highly sensitive to autoxidation. The reduction of inadvertently oxidized Corrinoids ([CoII]-state) is catalysed in an ATP-dependent reaction by RACE proteins, the reductive activators of Corrinoid-dependent enzymes. In this study, a reductive activator of O-demethylase Corrinoid proteins was characterized with respect to its ATPase and Corrinoid reduction activity. The reduction of the Corrinoid cofactor was dependent on the presence of potassium or ammonium ions. In the absence of the Corrinoid protein, a basal slow ATP hydrolysis was observed which was obviously not coupled to Corrinoid reduction. ATP hydrolysis was significantly stimulated by the Corrinoid protein in the [CoII]-state of the Corrinoid cofactor. The stoichiometry of ATP hydrolysed per mol Corrinoid reduced was near 1:1. Site-directed mutagenesis was applied to study the impact of a highly conserved region possibly involved in nucleotide binding of RACE proteins, indicating that an aspartate and a glycine residue may play an essential role for the function of the enzyme.
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Kinetic regulation of a Corrinoid‐reducing metallo‐ATPase by its substrates
Molecular microbiology, 2014Co-Authors: Martin Sperfeld, Gabriele Diekert, Sandra StudenikAbstract:Summary Corrinoid cofactors play a crucial role as methyl group carriers in the C1 metabolism of anaerobes, e.g. in the cleavage of phenyl methyl ethers by O-demethylases. For the methylation, the protein-bound Corrinoid has to be in the super-reduced [CoI]-state, which is highly sensitive to autoxidation. The reduction of inadvertently oxidized Corrinoids ([CoII]-state) is catalysed in an ATP-dependent reaction by RACE proteins, the reductive activators of Corrinoid-dependent enzymes. In this study, a reductive activator of O-demethylase Corrinoid proteins was characterized with respect to its ATPase and Corrinoid reduction activity. The reduction of the Corrinoid cofactor was dependent on the presence of potassium or ammonium ions. In the absence of the Corrinoid protein, a basal slow ATP hydrolysis was observed which was obviously not coupled to Corrinoid reduction. ATP hydrolysis was significantly stimulated by the Corrinoid protein in the [CoII]-state of the Corrinoid cofactor. The stoichiometry of ATP hydrolysed per mol Corrinoid reduced was near 1:1. Site-directed mutagenesis was applied to study the impact of a highly conserved region possibly involved in nucleotide binding of RACE proteins, indicating that an aspartate and a glycine residue may play an essential role for the function of the enzyme.
Joseph A Krzycki - One of the best experts on this subject based on the ideXlab platform.
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Kinetic and substrate complex characterization of RamA, a Corrinoid protein reductive activase from Methanosarcina barkeri.
FEMS microbiology letters, 2020Co-Authors: Katherine A. Huening, Ruisheng Jiang, Joseph A KrzyckiAbstract:In microbial Corrinoid-dependent methyltransferase systems, adventitious Co(I)-Corrinoid oxidation halts catalysis and necessitates repair by ATP-dependent reductive activases. RamA, an activase with a C-terminal ferredoxin domain with two [4Fe-4S] clusters from methanogenic archaea, has been far less studied than the bacterial activases bearing an N-terminal ferredoxin domain with one [2Fe-2S] cluster. These differences suggest RamA might prove to have other distinctive characteristics. Here, we examine RamA kinetics and the stoichiometry of the Corrinoid protein:RamA complex. Like bacterial activases, K+ stimulates RamA. Potassium stimulation had been questioned due to differences in the primary structure of bacterial and methanogen activases. Unlike one bacterial activase, ATP is not inhibitory allowing the first determination of apparent kinetic parameters for any Corrinoid activase. Unlike bacterial activases, a single RamA monomer complexes a single Corrinoid protein monomer. Alanine replacement of a RamA serine residue corresponding to the serine of one bacterial activase which ligates the Corrinoid cobalt during complex formation led to only moderate changes in the kinetics of RamA. These results reveal new differences in the two types of Corrinoid activases, and provide direct evidence for the proposal that Corrinoid activases act as catalytic monomers, unlike other enzymes that couple ATP hydrolysis to difficult reductions.
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RamA, a Protein Required for Reductive Activation of Corrinoid-dependent Methylamine Methyltransferase Reactions in Methanogenic Archaea
The Journal of biological chemistry, 2008Co-Authors: Tsuneo K. Ferguson, Jitesh A. Soares, Tanja Lienard, Gerhard Gottschalk, Joseph A KrzyckiAbstract:Archaeal methane formation from methylamines is initiated by distinct methyltransferases with specificity for monomethylamine, dimethylamine, or trimethylamine. Each methylamine methyltransferase methylates a cognate Corrinoid protein, which is subsequently demethylated by a second methyltransferase to form methyl-coenzyme M, the direct methane precursor. Methylation of the Corrinoid protein requires reduction of the central cobalt to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric iron-sulfur protein, was isolated from Methanosarcina barkeri and is required for in vitro ATP-dependent reductive activation of methylamine:CoM methyl transfer from all three methylamines. In the absence of the methyltransferases, highly purified RamA was shown to mediate the ATP-dependent reductive activation of Co(II) Corrinoid to the Co(I) state for the monomethylamine Corrinoid protein, MtmC. The ramA gene is located near a cluster of genes required for monomethylamine methyltransferase activity, including MtbA, the methylamine-specific CoM methylase and the pyl operon required for co-translational insertion of pyrrolysine into the active site of methylamine methyltransferases. RamA possesses a C-terminal ferredoxin-like domain capable of binding two tetranuclear iron-sulfur proteins. Mutliple ramA homologs were identified in genomes of methanogenic Archaea, often encoded near methyltrophic methyltransferase genes. RamA homologs are also encoded in a diverse selection of bacterial genomes, often located near genes for Corrinoid-dependent methyltransferases. These results suggest that RamA mediates reductive activation of Corrinoid proteins and that it is the first functional archetype of COG3894, a family of redox proteins of unknown function.
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the trimethylamine methyltransferase gene and multiple dimethylamine methyltransferase genes of methanosarcina barkeri contain in frame and read through amber codons
Journal of Bacteriology, 2000Co-Authors: Ligi Paul, Donald J Ferguson, Joseph A KrzyckiAbstract:The archaeal 16S rRNA tree has four major branches of methanogens, three of which make methane almost exclusively from carbon dioxide. A family of the fourth branch, the Methanosarcinaceae, is exceptional in that representative species such as Methanosarcina barkeri are also capable of methanogenesis from acetate or methylotrophic substrates, such as methanol, methylated thiols, and methylamines (3, 46). Methylamines are particularly important methane precursors in marine environments, where they arise from the breakdown of common osmolytes (22). Methanogenesis from trimethylamine (TMA) requires the intermediate formation of dimethylamine (DMA) and monomethylamine (MMA), which are subsequently converted to methane (16). The methylation of coenzyme M (CoM) with a methylamine initiates methanogenesis, and as with all substrates, methyl-CoM serves as the direct methane precursor (41, 47). The different pathways of TMA-, DMA-, and MMA-specific CoM methyl transfer can be reconstituted in vitro with only three highly purified polypeptides. A single protein, MtbA, acts as the common CoM methylase for all three methylamines (11). However, different methyltransferase polypeptides are required to initiate metabolism by demethylation of TMA, DMA, or MMA and subsequent methylation of different Corrinoid binding polypeptides. The methylated Corrinoid is then demethylated by MtbA to methylate CoM. Each gene or gene product involved in CoM methylation with a methylotrophic substrate is designated according to the following convention. The first two letters, mt, indicate involvement of the gene or gene product in methyl transfer. The third letter indicates the substrate: a for methanol, s for methylthiols, m for MMA, b for DMA, and t for TMA. The final letter designates the polypeptide function, where B is the substrate-specific methyltransferase that methylates the Corrinoid protein with substrate, C is the Corrinoid binding polypeptide, and A is the CoM-methylating protein. For MMA:CoM methyl transfer, the specific MMA methyltransferase is MtmB, which uses MMA to methylate its cognate Corrinoid protein, MtmC (5). For DMA:CoM methyl transfer, the specific DMA methyltransferase is MtbB (44, 45), which methylates the DMA Corrinoid protein, MtbC (D. J. Ferguson, N. Gorlatova, L. Paul, D. A. Grahame, and J. A. Krzycki, submitted for publication). The specific TMA methyltransferase, MttB, copurifies with the TMA Corrinoid protein, MttC. MttB has not yet been shown to directly methylate MttC with TMA. However, by analogy with the mechanism of CoM methylation with MMA or methanol, it was proposed that MttB is a TMA methyltransferase that uses TMA to methylate MttC (10). Figure Figure11 illustrates the functions of the gene products methylating CoM with either DMA or TMA. FIG. 1 Schematic of the mtt-mtb1 transcriptional unit. Above the gene sequence are indicated the reactions demonstrated for the gene products, i.e., the DMA and TMA methyltransferases and their cognate Corrinoid proteins. The function of mttP is proposed but ... Methanol:CoM methyl transfer also requires a specific methanol methyltransferase polypeptide, MtaB, which tightly binds and methylates its cognate Corrinoid protein, MtaC (7, 36). Methyl-MtaC is then demethylated by a different CoM methylase, MtaA, which methylates CoM. Methylthiol:CoM methyl transfer has been shown to require only two polypeptides (39, 40). In this case, a third CoM methylase, MtsA, appears to methylate a Corrinoid binding protein, MtsB, with methylated thiols like dimethylsulfide (MtsB is the only Corrinoid protein which is not named according to the above nomenclature rules). MtsA then demethylates methyl-MtsB and methylates CoM. The genes for MMA- (6), methanol- (35), and methylthiol-dependent (32) CoM methylation have been identified by reverse genetics. These studies have shown that the methylotrophic Corrinoid proteins MtaC, MtsB, and MtmC share approximately 50% identity. These methylotrophic Corrinoid proteins are also homologous to the cobalamin-binding domain of B12 proteins, such as methionine synthase (28). The methylcobamide-CoM methyltransferases, MtsA, MtaA, and MtbA, are also 50% similar to one another (14, 26, 32). However, MtaB and MtmB, the methanol and MMA methyltransferases, have no significant homology with one another. A surprising result from the sequencing of the gene encoding the MMA methyltransferase, MtmB, was the presence of a single in-frame amber codon midway through the open reading frame that does not function as a stop codon during translation of the mRNA producing the abundant full-length 50-kDa protein (6). The functionally analogous, but nonhomologous, methanol methyltransferase gene does not contain such an in-frame amber codon. Here, the genes encoding the TMA and DMA methyltransferases and their cognate Corrinoid proteins are characterized for the first time. Interestingly, single in-frame amber codons are found to be a common feature of the genes encoding the polypeptides that initiate methanogenesis from TMA, DMA, or MMA.
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Reconstitution of Monomethylamine:Coenzyme M methyl transfer with a Corrinoid protein and two methyltransferases purified from Methanosarcina barkeri.
The Journal of biological chemistry, 1997Co-Authors: Stephen Anthony Burke, Joseph A KrzyckiAbstract:Methyl transfer from dimethylamine to coenzyme M was reconstituted in vitro for the first time using only highly purified proteins. These proteins isolated from Methanosarcina barkeri included the previously unidentified Corrinoid protein MtbC, which copurified with MtbA, the methylCorrinoid:Coenzyme M methyltransferase specific for methanogenesis from methylamines. MtbC binds 1.0 mol of Corrinoid cofactor/mol of 24-kDa polypeptide and stimulated dimethylamine:coenzyme M methyl transfer 3.4-fold in a cell extract. Purified MtbC and MtbA were used to assay and purify a dimethylamine:Corrinoid methyltransferase, MtbB1. MtbB1 is a 230-kDa protein composed of 51-kDa subunits that do not possess a Corrinoid prosthetic group. Purified MtbB1, MtbC, and MtbA were the sole protein requirements for in vitro dimethylamine:coenzyme M methyl transfer. An MtbB1:MtbC ratio of 1 was optimal for coenzyme M methylation with dimethylamine. MtbB1 methylated either Corrinoid bound to MtbC or free cob(I)alamin with dimethylamine, indicating MtbB1 carries an active site for dimethylamine demethylation and Corrinoid methylation. Experiments in which different proteins of the resolved monomethylamine:coenzyme M methyl transfer reaction replaced proteins involved in dimethylamine:coenzyme M methyl transfer indicated high specificity of MtbB1 and MtbC in dimethylamine:coenzyme M methyl transfer activity. These results indicate MtbB1 demethylates dimethylamine and specifically methylates the Corrinoid prosthetic group of MtbC, which is subsequently demethylated by MtbA to methylate coenzyme M during methanogenesis from dimethylamine.
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Coenzyme M methylase activity of the 480-kilodalton Corrinoid protein from Methanosarcina barkeri.
Journal of bacteriology, 1996Co-Authors: Thomas C. Tallant, Joseph A KrzyckiAbstract:Activity staining of extracts of Methanosarcina barkeri electrophoresed in polyacrylamide gels revealed an additional methylcobalamin:coenzyme M (methylcobalamin:CoM) methyltransferase present in cells grown on acetate but not in those grown on trimethylamine. This methyltransferase is the 480-kDa Corrinoid protein previously identified by its methylation following inhibition of methyl-CoM reductase in otherwise methanogenic cell extracts. The methylcobalamin:CoM methyltransferase activity of the purified 480-kDa protein increased from 0.4 to 3.8 micromol/min/mg after incubation with sodium dodecyl sulfate (SDS). Following SDS-polyacrylamide gel electrophoresis analysis of unheated protein samples, a polypeptide with an apparent molecular mass of 48 kDa which possessed methylcobalamin:CoM methyltransferase activity was detected. This polypeptide migrated with an apparent mass of 41 kDa when the 480-kDa protein was heated before electrophoresis, indicating that the alpha subunit is responsible for the activity. The N-terminal sequence of this subunit was 47% similar to the N termini of the A and M isozymes of methylcobalamin:CoM methyltransferase (methyltransferase II). The endogenous methylated Corrinoid bound to the beta subunit of the 480-kDa protein could be demethylated by CoM, but not by homocysteine or dithiothreitol, resulting in a Co(I) Corrinoid. The Co(I) Corrinoid could be remethylated by methyl iodide, and the protein catalyzed a methyl iodide:CoM transmethylation reaction at a rate of 2.3 micromol/min/mg. Methyl-CoM was stoichiometrically produced from CoM, as demonstrated by high-pressure liquid chromatography with indirect photometric detection. Two thiols, 2-mercaptoethanol and mercapto-2-propanol, were poorer substrates than CoM, while several others tested (including 3-mercaptopropanesulfonate) did not serve as methyl acceptors. These data indicate that the 480-kDa Corrinoid protein is composed of a novel isozyme of methyltransferase II which remains firmly bound to a Corrinoid cofactor binding subunit during isolation.
Bernhard Krautler - One of the best experts on this subject based on the ideXlab platform.
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exogenous 5 6 dimethylbenzimidazole caused production of a non functional tetrachloroethene reductive dehalogenase in sulfurospirillum multivorans
Environmental Microbiology, 2014Co-Authors: Sebastian Keller, Bernhard Krautler, Gabriele Diekert, Markus Ruetz, Cindy Kunze, Torsten SchubertAbstract:Summary Corrinoid-dependent reductive dehalogenation is mediated by phylogenetically diverse anaerobic bacteria that either synthesize Corrinoids de novo or are dependent on Corrinoid salvaging from the environment. The tetrachloroethene (PCE) reduc- tive dehalogenase (PceA) of the Gram-negative Epsilonproteobacterium Sulfurospirillum multivorans harbours a norpseudo-B12 as Corrinoid cofactor. Norpseudo-B12 differs from coenzyme B12 in the nucleotide loop structure. Adenine instead of 5,6- dimethylbenzimidazole (DMB) serves as lower ligand base of the central cobalt ion, and the nucleotide loop of norpseudo-B12 lacks a methyl group at position 176. In this study, S. multivorans was grown anaerobically with PCE in the presence of DMB. At a DMB concentration of 25 M, the adenine moiety in the nucleotide loop of norpseudo-B12 was quantita- tively replaced by DMB. The formation of the DMB- containing nor-B12 severely affected PCE-dependent growth and the PceA activity. In DMB-treated cells processing of the cytoplasmic PceA precursor was impeded, a result pointing to retarded cofactor incorporation. PceA enriched from cells cultivated with DMB contained nor-B12. Nor-B12 purified from cells grown in the presence of DMB mediated the abiotic reductive dehalogenation of trichloroacetate to dichloroacetate at a 25-fold lower rate in compari-
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a blue Corrinoid from partial degradation of vitamin b12 in aqueous bicarbonate spectra structure and interaction with proteins of b12 transport
Biochemistry, 2011Co-Authors: Sergey N Fedosov, Markus Ruetz, Karl Gruber, Natalya U Fedosova, Bernhard KrautlerAbstract:Cobalamin (Cbl) is a complex cofactor produced only by bacteria but used by all animals and humans. Cyanocobalamin (vitamin B12, CNCbl) is one commonly isolated form of cobalamin. B12 belongs to a large group of Corrinoids, which are characterized by a distinct red color conferred by the system of conjugated double bonds of the corrin ring retaining a Co(III) ion. A unique blue Cbl derivative was produced by hydrolysis of CNCbl in a weakly alkaline aqueous solution of bicarbonate. This Corrinoid was purified and isolated as dark blue crystals. Its spectroscopic analysis and X-ray crystallography revealed B-ring opening with formation of 7,8-seco-cyanocobalamin (7,8-sCNCbl). The unprecedented structural change was caused by cleavage of the peripheral C–C bond between saturated carbons 7 and 8 of the corrin macrocycle accompanied by formation of a C═C bond at C7 and a carbonyl group at C8. Additionally, the C-amide was hydrolyzed to a carboxylic acid. The extended conjugation of the π-system caused a consid...
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Encyclopedia of Catalysis - Cobalt Enzymes/Models
Encyclopedia of Catalysis, 2010Co-Authors: Bernhard KrautlerAbstract:Vitamin B12 and its coenzyme forms are prominent cobalt-containing cofactors and part of the larger class of the natural tetrapyrrolic metal complexes. Such cobalt Corrinoids are an important component of human nutrition, and many (other) forms of life also depend upon them. Cobalt-containing Corrinoid cofactors play basic roles in carbon fixation and energy metabolism in anaerobes, as well as in the carbon metabolism in a broad range of lower organisms. Microorganisms developed unique B12 biosynthetic means and, indeed, are the only natural sources of the B12 derivatives. Other organisms have evolved intricate strategies for the uptake and transport of the Corrinoids. The cofactor functions of cobalt Corrinoids are a result of their extraordinary organometallic chemistry under physiological conditions and their redox chemistry. Coenzyme B12 (a reversible source of a free radical), methylcobalamin (a versatile methylating agent), and reduced (supernucleophilic or radicaloid) Corrinoids are cofactors in a range of enzymes. These enzymes channel the specific reactivity of B12 cofactors into biological catalysis of unique reactions, involving radicals and organometallic intermediates. B12-dependent enzymes thus pose still puzzling structural and mechanistic questions, as does the recently discovered interaction of B12 cofactors with RNA. Keywords: bioorganometallic chemistry; cofactor; methyl transfer; radical enzymes; vitamin B12
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Adenosyl-176-norcobinamide – A likely biosynthetic precursor to natural 176-norvitamin B12 derivatives
Journal of Organometallic Chemistry, 2007Co-Authors: Philip A. Butler, Bernhard KrautlerAbstract:The “complete” Corrinoid 176-norpseudovitamin B12 was recently isolated as the cyano-Co(III)-form of the Corrinoid cofactor of tetrachlorethene reductive dehalogenase of the anaerobe Sulfurospirillum (formerly Dehalospirillum) multivorans. 176-Norpseudovitamin B12 represents the first example of (the cyano-Co(III)-form of) a naturally occurring “complete” B12 cofactor lacking a characteristic peripheral methyl group of the cobamide ligand. Its discovery has generated interest in 176-nor-B12 derivatives, “complete” Corrinoids lacking the methyl group attached to carbon 176. Here, we report the preparation of Coβ-5′-adenosyl-176-norcobinamide by in situ alkylation of Co(I)-176-norcobinamide, obtained from electrochemical reduction of Coα,Coβ-dicyano-176-norcobinamide. Since Coβ-5′-adenosylcobinamide is a biosynthetic intermediate of the complete cobamides, Coβ-5′-adenosyl-176-norcobinamide is a “rational” biosynthetic precursor for natural 176-nor-B12 derivatives. The spectroscopic data for adenosyl-176-norcobinamide establish the suggested structure of the title compound and give further evidence for the extensive flexibility and conformational dynamics of the organometallic 5′-deoxy-5′-adenosyl ligand.
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The Corrinoid from Methanobacterium thermoautotrophicum (Marburg strain)
FEBS Journal, 2005Co-Authors: Bernhard Krautler, Johanna Moll, Rudolf K. ThauerAbstract:The Corrinoids from Methanobacterium thermoautotrophicum were extracted as the Co-cyano derivative, which was isolated in crystalline form. A consistent set of spectroscopic data was acquired (ultraviolet/visible, circular dichroic, infrared, fast-atom-bombardment mass, 1H-NMR and 13C-NMR spectra), which allowed the structural analysis of this complete Corrinoid. It was assigned the structure of the Coβ-cyano-5′-hydroxybenzimidazolylcobamide and was identified with Friedrich and Bernhauer's ‘factor III’ by comparison with an authentic sample.
Sandra Studenik - One of the best experts on this subject based on the ideXlab platform.
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Flavodoxin hydroquinone provides electrons for the ATP-dependent reactivation of protein-bound Corrinoid cofactors.
The FEBS journal, 2020Co-Authors: Lena Kißling, Hendrike Dürichen, Yvonne Greiser, Sandra StudenikAbstract:Corrinoid-dependent enzyme systems rely on the super-reduced state of the protein-bound Corrinoid cofactor to be functional, for example, in methyl transfer reactions. Due to the low redox potential of the [CoII ]/[CoI ] couple, autoxidation of the Corrinoid cofactor occurs and leads to the formation of the inactive [CoII ]-state. For the reactivation, which is an energy-demanding process, electrons have to be transferred from a physiological donor to the Corrinoid cofactor by the help of a reductive activator protein. In this study, we identified reduced flavodoxin as electron donor for the ATP-dependent reduction of protein-bound Corrinoid cofactors of bacterial O-demethylase enzyme systems. Reduced flavodoxin was generated enzymatically using pyruvate:ferredoxin/flavodoxin oxidoreductase rather than hydrogenase. Two of the four flavodoxins identified in Acetobacterium dehalogenans and Desulfitobacterium hafniense DCB-2 were functional in supplying electrons for Corrinoid reduction. They exhibited a midpoint potential of about -400 mV (ESHE , pH 7.5) for the semiquinone/hydroquinone transition. Reduced flavodoxin could be replaced by reduced clostridial ferredoxin. It was shown that the low-potential electrons of reduced flavodoxin are first transferred to the iron-sulfur cluster of the reductive activator and finally to the protein-bound Corrinoid cofactor. This study further highlights the importance of reduced flavodoxin, which allows maintaining a variety of enzymatic reaction cycles by delivering low-potential electrons.
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Redox potential changes during ATP‐dependent Corrinoid reduction determined by redox titrations with europium(II)–DTPA
Protein science : a publication of the Protein Society, 2019Co-Authors: Hendrike Dürichen, Gabriele Diekert, Sandra StudenikAbstract:Corrinoids are essential cofactors of enzymes involved in the C1 metabolism of anaerobes. The active, super-reduced [CoI ] state of the Corrinoid cofactor is highly sensitive to autoxidation. In O-demethylases, the oxidation to inactive [CoII ] is reversed by an ATP-dependent electron transfer catalyzed by the activating enzyme (AE). The redox potential changes of the Corrinoid cofactor, which occur during this reaction, were studied by potentiometric titration coupled to UV/visible spectroscopy. By applying europium(II)-diethylenetriaminepentaacetic acid (DTPA) as a reductant, we were able to determine the midpoint potential of the [CoII ]/[CoI ] couple of the protein-bound Corrinoid cofactor in the absence and presence of AE and/or ATP. The data revealed that the transfer of electrons from a physiological donor to the Corrinoid as the electron-accepting site is achieved by increasing the potential of the Corrinoid cofactor from -530 ± 15 mV to -250 ± 10 mV (ESHE , pH 7.5). The first 50 to 100 mV of the shift of the redox potential seem to be caused by the interaction of nucleotide-bound AE with the Corrinoid protein or its cofactor. The remaining 150-200 mV had to be overcome by the chemical energy of ATP hydrolysis. The experiments revealed that Eu(II)-DTPA, which was already known as a powerful reducing agent, is a suitable electron donor for titration experiments of low-potential redox centers. Furthermore, the results of this study will contribute to the understanding of thermodynamically unfavorable electron transfer processes driven by the power of ATP hydrolysis.
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redox potential changes during atp dependent Corrinoid reduction determined by redox titrations with europium ii dtpa
Protein Science, 2019Co-Authors: Hendrike Dürichen, Gabriele Diekert, Sandra StudenikAbstract:Corrinoids are essential cofactors of enzymes involved in the C1 metabolism of anaerobes. The active, super-reduced [CoI ] state of the Corrinoid cofactor is highly sensitive to autoxidation. In O-demethylases, the oxidation to inactive [CoII ] is reversed by an ATP-dependent electron transfer catalyzed by the activating enzyme (AE). The redox potential changes of the Corrinoid cofactor, which occur during this reaction, were studied by potentiometric titration coupled to UV/visible spectroscopy. By applying europium(II)-diethylenetriaminepentaacetic acid (DTPA) as a reductant, we were able to determine the midpoint potential of the [CoII ]/[CoI ] couple of the protein-bound Corrinoid cofactor in the absence and presence of AE and/or ATP. The data revealed that the transfer of electrons from a physiological donor to the Corrinoid as the electron-accepting site is achieved by increasing the potential of the Corrinoid cofactor from -530 ± 15 mV to -250 ± 10 mV (ESHE , pH 7.5). The first 50 to 100 mV of the shift of the redox potential seem to be caused by the interaction of nucleotide-bound AE with the Corrinoid protein or its cofactor. The remaining 150-200 mV had to be overcome by the chemical energy of ATP hydrolysis. The experiments revealed that Eu(II)-DTPA, which was already known as a powerful reducing agent, is a suitable electron donor for titration experiments of low-potential redox centers. Furthermore, the results of this study will contribute to the understanding of thermodynamically unfavorable electron transfer processes driven by the power of ATP hydrolysis.
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kinetic regulation of a Corrinoid reducing metallo atpase by its substrates
Molecular Microbiology, 2014Co-Authors: Martin Sperfeld, Gabriele Diekert, Sandra StudenikAbstract:Summary Corrinoid cofactors play a crucial role as methyl group carriers in the C1 metabolism of anaerobes, e.g. in the cleavage of phenyl methyl ethers by O-demethylases. For the methylation, the protein-bound Corrinoid has to be in the super-reduced [CoI]-state, which is highly sensitive to autoxidation. The reduction of inadvertently oxidized Corrinoids ([CoII]-state) is catalysed in an ATP-dependent reaction by RACE proteins, the reductive activators of Corrinoid-dependent enzymes. In this study, a reductive activator of O-demethylase Corrinoid proteins was characterized with respect to its ATPase and Corrinoid reduction activity. The reduction of the Corrinoid cofactor was dependent on the presence of potassium or ammonium ions. In the absence of the Corrinoid protein, a basal slow ATP hydrolysis was observed which was obviously not coupled to Corrinoid reduction. ATP hydrolysis was significantly stimulated by the Corrinoid protein in the [CoII]-state of the Corrinoid cofactor. The stoichiometry of ATP hydrolysed per mol Corrinoid reduced was near 1:1. Site-directed mutagenesis was applied to study the impact of a highly conserved region possibly involved in nucleotide binding of RACE proteins, indicating that an aspartate and a glycine residue may play an essential role for the function of the enzyme.
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Kinetic regulation of a Corrinoid‐reducing metallo‐ATPase by its substrates
Molecular microbiology, 2014Co-Authors: Martin Sperfeld, Gabriele Diekert, Sandra StudenikAbstract:Summary Corrinoid cofactors play a crucial role as methyl group carriers in the C1 metabolism of anaerobes, e.g. in the cleavage of phenyl methyl ethers by O-demethylases. For the methylation, the protein-bound Corrinoid has to be in the super-reduced [CoI]-state, which is highly sensitive to autoxidation. The reduction of inadvertently oxidized Corrinoids ([CoII]-state) is catalysed in an ATP-dependent reaction by RACE proteins, the reductive activators of Corrinoid-dependent enzymes. In this study, a reductive activator of O-demethylase Corrinoid proteins was characterized with respect to its ATPase and Corrinoid reduction activity. The reduction of the Corrinoid cofactor was dependent on the presence of potassium or ammonium ions. In the absence of the Corrinoid protein, a basal slow ATP hydrolysis was observed which was obviously not coupled to Corrinoid reduction. ATP hydrolysis was significantly stimulated by the Corrinoid protein in the [CoII]-state of the Corrinoid cofactor. The stoichiometry of ATP hydrolysed per mol Corrinoid reduced was near 1:1. Site-directed mutagenesis was applied to study the impact of a highly conserved region possibly involved in nucleotide binding of RACE proteins, indicating that an aspartate and a glycine residue may play an essential role for the function of the enzyme.
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substrate and cofactor reactivity of a carbon monoxide dehydrogenase Corrinoid enzyme complex stepwise reduction of iron sulfur and Corrinoid centers the Corrinoid cobalt 2 1 redox midpoint potential and overall synthesis of acetyl coa
Biochemistry, 1993Co-Authors: David A GrahameAbstract:: Cleavage of the acetyl carbon-carbon bond of acetyl-CoA in Methanosarcina barkeri is catalyzed by a high molecular mass multienzyme complex. The complex contains a Corrinoid protein and carbon monoxide dehydrogenase and requires tetrahydrosarcinapterin (H4SPt) as methyl group acceptor. Reactions of the enzyme complex with carbon monoxide and with the methyl group donor N5-methyltetrahydrosarcinapterin (CH3-H4SPt) have been analyzed by UV-visible spectroscopy. Reduction of the enzyme complex by CO occurred in two steps. In the first step, difference spectra exhibited peaks of maximal absorbance decrease at 426 nm (major) and 324 nm (minor), characteristic of Fe-S cluster reduction. In the second step, Corrinoid reduction to the Co1+ level was indicated by a prominent peak of increased absorbance at 394 nm. Spectrophotometric analyses of the Corrinoid redox state were performed on the intact complex at potentials poised by equilibration with gas mixtures containing different [CO2]/[CO] ratios or by variation of the [H+]/[H2] ratio. The Corrinoid Co2+/1+ midpoint potential was -426 mV (+/- 4 mV, n = 1.16 electrons, 24 degrees C), independent of pH (pH 6.4-8.0). The results indicated a significant fraction of Co1+ Corrinoid at potentials existing in vivo. The reduced Corrinoid reacted very rapidly with CH3-H4SPt. Reaction with methyl iodide was slow, and methylation by S-adenosylmethionine was not observed. Tne rate of methyl group transfer from CH3-H4SPt greatly exceeded the rate of CO reduction of enzyme centers. The enzyme complex catalyzed efficient synthesis of acetyl-CoA from coenzyme A, CO, and CH3-H4SPt. During acetyl-CoA synthesis, demethylation of CH3-H4SPt was monitored by the absorbance increase at 312 nm.(ABSTRACT TRUNCATED AT 250 WORDS)
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Substrate and cofactor reactivity of a carbon monoxide dehydrogenase-Corrinoid enzyme complex: Stepwise reduction of iron-sulfur and Corrinoid centers, the Corrinoid cobalt(2+/1+) redox midpoint potential, and overall synthesis of acetyl-CoA
Biochemistry, 1993Co-Authors: David A GrahameAbstract:Cleavage of the acetyl carbon-carbon bond of acetyl-CoA in Methanosarcina barkeri is catalyzed by a high molecular mass multienzyme complex. The complex contains a Corrinoid protein and carbon monoxide dehydrogenase and requires tetrahydrosarcinapterin (H4SPt) as methyl group acceptor. Reactions of the enzyme complex with carbon monoxide and with the methyl group donor N5-methyltetrahydrosarcinapterin (CH3-H4SPt) have been analyzed by UV-visible spectroscopy. Reduction of the enzyme complex by CO occurred in two steps. In the first step, difference spectra exhibited peaks of maximal absorbance decrease at 426 nm (major) and 324 nm (minor), characteristic of Fe-S cluster reduction. In the second step, Corrinoid reduction to the Co1+ level was indicated by a prominent peak of increased absorbance at 394 nm. Spectrophotometric analyses of the Corrinoid redox state were performed on the intact complex at potentials poised by equilibration with gas mixtures containing different [CO2]/[CO] ratios or by variation of the [H+]/[H2] ratio. The Corrinoid Co2+/1+ midpoint potential was -426 mV (+/- 4 mV, n = 1.16 electrons, 24 degrees C), independent of pH (pH 6.4-8.0). The results indicated a significant fraction of Co1+ Corrinoid at potentials existing in vivo. The reduced Corrinoid reacted very rapidly with CH3-H4SPt. Reaction with methyl iodide was slow, and methylation by S-adenosylmethionine was not observed. Tne rate of methyl group transfer from CH3-H4SPt greatly exceeded the rate of CO reduction of enzyme centers. The enzyme complex catalyzed efficient synthesis of acetyl-CoA from coenzyme A, CO, and CH3-H4SPt. During acetyl-CoA synthesis, demethylation of CH3-H4SPt was monitored by the absorbance increase at 312 nm.(ABSTRACT TRUNCATED AT 250 WORDS)
