Coenzyme B induced coordination of coenzyme M via its thiol group to Ni(I) of F430 in active methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) catalyzes the reaction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. At the active site, it contains the nickel porphinoid F430, which has to be in the Ni(I) oxidation state for the enzyme to be active. How the substrates interact with the active site Ni(I) has remained elusive. We report here that coenzyme M (HS-CoM), which is a reversible competitive inhibitor to methyl-coenzyme M, interacts with its thiol group with the Ni(I) and that for interaction the simultaneous presence of coenzyme B is required. The evidence is based on X-band continuous wave EPR and Q-band hyperfine sublevel correlation spectroscopy of MCR in the red2 state induced with 33S-labeled coenzyme M and unlabeled coenzyme B.     

On the mechanism of methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) catalyzes the reaction of methyl-coenzyme M (CH3-SCoM) and coenzyme B (HS-CoB) to methane and the corresponding heterodisulfide CoM-S-S-CoB. This unique reaction proceeds under strictly anaerobic conditions in the presence of coenzyme F430, a Ni-porphinoid. MCR is a large (alphabetagamma)2 heterohexameric protein complex containing two 50 A long active sites channels. Coenzyme F430 is embedded at the channel bottom and the substrates CH3-SCoM and HS-CoB bind in front of F430 into a solvent free and hydrophobic channel segment. Two principally different catalytic mechanisms are currently discussed. Mechanism I is based on a nucleophilic attack of Ni(I) onto the methyl group of CH3-SCoM yielding methyl-Ni(III) and mechanism II on an attack of Ni(I) onto the thioether sulfur of CH3-SCoM generating a Ni(II)-SCoM intermediate. Both mechanisms are discussed in the light of a large number of data collected about MCR over the last twenty years.     

Direct determination of the number of electrons needed to reduce coenzyme F430 pentamethyl ester to the Ni(I) species exhibiting the electron paramagnetic resonance and ultraviolet-visible spectra characteristic for the MCR(red1) state of methyl-coenzyme M reductase

The UV-visible and electron paramagnetic resonance (EPR) spectra of MCR(red1), the catalytically active state of methyl-coenzyme M reductase, are almost identical to those observed when free coenzyme F430 or its pentamethyl ester (F430M) are reduced to the Ni(I) valence state. Investigations and proposals concerning the catalytic mechanism of MCR were therefore based on MCR(red1) containing Ni(I)F430 until, in a recent report, Tang et al. (J. Am. Chem. Soc. 2002, 124, 13242) interpreted their resonance Raman data and titration experiments as indicating that, in MCR(red1), coenzyme F430 is not only reduced at the nickel center but at one of the C=N double bonds of the hydrocorphinoid macrocycle as well. To resolve this contradiction, we have investigated the stoichiometry of the reduction of coenzyme F430 pentamethyl ester (F430M) by three independent methods. Spectroelectrochemistry showed clean reduction to a single product that exhibits the UV-vis spectrum typical for MCR(red1). In three bulk electrolysis experiments, 0.96 +/- 0.1 F/mol was required to generate the reduced species. Reduction with decamethylcobaltocene in tetrahydrofuran (THF) consumed 1 mol of (Cp)(2)Co/mol of F430M, and the stoichiometry of the reoxidation of the reduced form with the two-electron oxidant methylene blue was 0.46 +/- 0.05 mol of methylene blue/mol of reduced F430M. These experiments demonstrate that the reduction of coenzyme F430M to the species having almost identical UV-vis and EPR spectra as MCR(red1) is a one-electron process and therefore inconsistent with a reduction of the macrocycle chromophore.     

Cryoreduction of methyl-coenzyme M reductase: EPR characterization of forms, MCR(ox1) and MCR (red1).

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methyl-coenzyme M (CH(3)S-CH(2)CH(2)SO(3)) from methane. The active site is a nickel tetrahydrocorphinoid cofactor, factor 430, which in inactive form contains EPR-silent Ni(II). Two such forms, denoted MCR(silent) and MCR(ox1)(-)(silent), were previously structurally characterized by X-ray crystallography. We describe here the cryoreduction of both of these MCR forms by gamma-irradiation at 77 K, which yields reduced protein maintaining the structure of the oxidized starting material. Cryoreduction of MCR(silent) yields an EPR signal that strongly resembles that of MCR(red1), the active form of MCR; and stepwise annealing to 260-270 K leads to formation of MCR(red1). Cryoreduction of MCR(ox1)(-)(silent) solutions shows that our preparative method for this state yields enzyme that contains two major forms. One behaves similarly to MCR(silent), as shown by the observation that both of these forms give essentially the same redlike EPR signals upon cryoreduction, both of which give MCR(red1) upon annealing. The other form is assigned to the crystallographically characterized MCR(ox1)(-)(silent) and directly gives MCR(ox1) upon cryoreduction. X-band spectra of these cryoreduced samples, and of conventionally prepared MCR(red1) and MCR(ox1), all show resolved hyperfine splitting from four equivalent nitrogen ligands with coupling constants in agreement with those determined in previous EPR studies and from (14)N ENDOR of MCR(red1) and MCR(ox1). These experiments have confirmed that all EPR-visible forms of MCR contain Ni(I) and for the first time generated in vitro the EPR-visible, enzymatically active MCR(red1) and the activate-able "ready" MCR(ox1) from "silent" precursors. Because the solution Ni(II) species we assign as MCR(ox1)(-)(silent) gives as its primary cryoreduction product the Ni(I) state MCR(ox1), previous crystallographic data on MCR(ox1)(-)(silent) allow us to identify the exogenous axial ligand in MCR(ox1) as the thiolate from CoM; the cryoreduction experiments further allow us to propose possible axial ligands in MCR(red1). The availability of model compounds for MCR(red1) and MCR(ox1) also is discussed.     

X-ray absorption and resonance Raman studies of methyl-coenzyme M reductase indicating that ligand exchange and macrocycle reduction accompany reductive activation

Methyl-coenzyme M reductase (MCR) catalyzes methane formation from methyl-coenzyme M (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoBSH). MCR contains a nickel hydrocorphin cofactor at its active site, called cofactor F(430). Here we present evidence that the macrocyclic ligand participates in the redox chemistry involved in catalysis. The active form of MCR, the red1 state, is generated by reducing another spectroscopically distinct form called ox1 with titanium(III) citrate. Previous electron paramagnetic resonance (EPR) and (14)N electron nuclear double resonance (ENDOR) studies indicate that both the ox1 and red1 states are best described as formally Ni(I) species on the basis of the character of the orbital containing the spin in the two EPR-active species. Herein, X-ray absorption spectroscopic (XAS) and resonance Raman (RR) studies are reported for the inactive (EPR-silent) forms and the red1 and ox1 states of MCR. RR spectra are also reported for isolated cofactor F(430) in the reduced, resting, and oxidized states; selected RR data are reported for the (15)N and (64)Ni isotopomers of the cofactor, both in the intact enzyme and in solution. Small Ni K-edge energy shifts indicate that minimal electron density changes occur at the Ni center during redox cycling of the enzyme. Titrations with Ti(III) indicate a 3-electron reduction of free cofactor F(430) to generate a stable Ni(I) state and a 2-electron reduction of Ni(I)-ox1 to Ni(I)-red1. Analyses of the XANES and EXAFS data reveal that both the ox1 and red1 forms are best described as hexacoordinate and that the main difference between ox1 and red1 is the absence of an axial thiolate ligand in the red1 state. The RR data indicate that cofactor F(430) undergoes a significant conformational change when it binds to MCR. Furthermore, the vibrational characteristics of the ox1 state and red1 states are significantly different, especially in hydrocorphin ring modes with appreciable C=N stretching character. It is proposed that these differences arise from a 2-electron reduction of the hydrocorphin ring upon conversion to the red1 form. Presumably, the ring-reduction and ligand-exchange reactions reported herein underlie the enhanced activity of MCR(red1), the only form of MCR that can react productively with the methyl group of methyl-SCoM.     

A new mechanism for methane production from methyl-coenzyme M reductase as derived from density functional calculations

We propose a new DFT-based mechanism for methane production using the full F430 cofactor of MCR (methyl-coenzyme M reductase) along with a coordinated O=CH2CH2C(H)NH2C(H)O (surrogate for glutamine) as a model of the active site for conversion of CH3SCoM(-) (CH3SCH2CH2SO3(-)) + HSCoB to methane plus the corresponding heterodisulfide. The cycle begins with the protonation of F430, either on Ni or on the C-ring nitrogen of the tetrapyrrole ring, both of which are nearly equally favorable. The C-ring protonated form is predicted to oxidatively add CH3SCoM(-) to give a 4-coordinate Ni center where the Ni moves out of the plane of the four ring nitrogens. The movement of Ni (and the attached CH3 and SCH2CH2SO3(2-) ligands) toward the SCoB(-) (deprotonated HSCoB) cofactor allows a 2c-3e interaction to form between the two sulfur atoms. The release of the heterodisulfide yields a Ni(III) center with a methyl group attached. The concerted elimination of methane, where the methyl group coordinated to Ni abstracts the proton from the C-ring nitrogen, has a very small calculated activation barrier (5.4 kcal/mol). The NPA charge on Ni for the various reaction steps indicates that the oxidation state changes occur largely on the attached ligands.     

Rapid ligand exchange in the MCRred1 form of methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) from Methanothermobacter marburgensis (Mtm), catalyses the final step in methane synthesis in all methanogenic organisms. Methane is produced by coenzyme B-dependent two-electron reduction of methyl-coenzyme M. At the active site of MCR is the corphin cofactor F(430), which provides four-coordination through the pyrrole nitrogens to a central Ni ion in all states of the enzyme. The important MCRox1 ("ready") and MCRred1 ("active") states contain six-coordinate Ni(I) and differ in their upper axial ligands; furthermore, red1 appears to be two-electrons more reduced than in ox1 and other Ni(II) states that have been studied. On the basis of the reactivity of MCRred1 and MCRox1 with a substrate analogue and inhibitor (3-bromopropanesulfonate) and other small molecules (chloroform, dichloromethane, mercaptoethanol, and nitric oxide), we present evidence that the six-coordinate Ni(I) centers in the MCRred1 and MCRox1 states exhibit markedly different inherent reactivities. MCRred1 reacts faster with chloroform (2100-fold or 35000-fold when corrected for temperature effects), nitric oxide (90-fold), and 3-bromopropanesulfonate (10(6)-fold) than MCRox1. MCRred1 reacts with chloroform and dichloromethane and, like F(430), can catalyze dehalogenation reactions and produce lower halogenated products. We conclude that the enhanced reactivity of MCRred1 is due to the replacement of a relatively exchange-inert thiol ligand in MCRox1 with a weakly coordinating upper axial ligand in red1 that can be easily replaced by incoming ligands.     

Model studies of methyl CoM reductase: methane formation via CH3-S bond cleavage of Ni(I) tetraazacyclic complexes having intramolecular methyl sulfide pendants

The Ni(I) tetraazacycles [Ni(dmmtc)](+) and [Ni(mtc)](+), which have methylthioethyl pendants, were synthesized as models of the reduced state of the active site of methyl coenzyme M reductase (MCR), and their structures and redox properties were elucidated (dmmtc, 1,8-dimethyl-4,11-bis{(2-methylthio)ethyl}-1,4,8,11-tetraaza-1,4,8,11-cyclotetradecane; mtc, 1,8-{bis(2-methylthio)ethyl}-1,4,8,11-tetraaza-1,4,8,11-cyclotetradecane). The intramolecular CH(3)-S bond of the thioether pendant of [Ni(I)(dmmtc)](OTf) was cleaved in THF at 75 °C in the presence of the bulky thiol DmpSH, which acts as a proton source, and methane was formed in 31% yield and a Ni(II) thiolate complex was concomitantly obtained (Dmp = 2,6-dimesityphenyl). The CH(3)-S bond cleavage of [Ni(I)(mtc)](+) also proceeded similarly, but under milder conditions probably due to the lower potential of the [Ni(I)(mtc)](+) complex. These results indicate that the robust CH(3)-S bond can be homolytically cleaved by the Ni(I) center when they are properly arranged, which highlights the significance of the F430 Ni environment in the active site of the MCR protein.     

Effect of the methyl-coenzyme-m reductase protein matrix on the hole-size and nonplanar deformations of coenzyme F430

Methyl-coenzyme-M reductase (MCR) is a key enzyme common to all methane-producing pathogens. It catalyses the final step in methane synthesis. Each MCR contains two noncovalently bound molecules of cofactor F430. Normal-coordinate structural decomposition, hole-size analysis, and molecular mechanics calculations were undertaken to examine the effect of MCR on the hole-size and nonplanar deformations of coenzyme F430. In MCR, the protein prevents F430 from undergoing nonplanar deformations, which results in a more rigid tetrahydrocorphinoid cofactor that has a shorter ideal metal-nitrogen distance in the MCR protein matrix than it does in solution. Changing the coordination number of the nickel ion in F430 has a very small effect on the ideal hole size; however, it has a significant effect on the nonplanar deformations the coenzyme undergoes upon contraction and expansion. In all complexes we examined, cofactor F430 undergoes more nonplanar deformations when it contains a four-coordinate metal ion than it does when it contains a six-coordinate metal ion. Clearly, MCR moderates the hole-size and the nonplanar deformations of coenzyme F430, which are known to affect redox potentials and axial ligand affinities. This suggests that the protein environment may be responsible for tuning the chemistry of the active-site nickel ion.     

Moderating influence of proteins on nonplanar tetrapyrrole deformations: coenzyme F430 in methyl-coenzyme-M reductase

Normal-coordinate structural decomposition, cluster analysis, and molecular mechanics calculations were undertaken to examine the effect of methyl-coenzyme-M reductase (MCR) on the nonplanar deformations of coenzyme F430. Although free 12,13-diepi-F430 has a lower energy conformation than free F430, the protein restraints exerted by MCR are responsible for F430 having a lower energy conformation than the 12,13-diepimer in MCR. According to the NSD analysis, the crystal structure of free diepimerized F430M is highly distorted. In MCR the protein prevents 12,13-diepi-F430 from undergoing nonplanar deformations; therefore, MCR favors F430 over the 12,13-diepimeric form. The strain imposed on 12,13-diepi-F430 in the protein is so large that although 88% of free F430 is found in the diepimeric form, none of the diepimeric form is found in MCR. This is of significance since the two forms have different chemistries. MCR also moderates the nonplanar deformations of coenzyme F430, which are known to affect redox potentials and axial ligand affinities in tetrapyrroles, suggesting that the protein environment (MCR) is responsible for tuning the chemistry of the active site nickel ion. F430 is bound to MCR by hydrogen bonds between the protein and the F430 carboxylate groups. Conformational searches have shown that F430 has very little rotational and translational freedom within MCR.     

Structural analysis of a Ni-methyl species in methyl-coenzyme M reductase from Methanothermobacter marburgensis

We present the 1.2 ? resolution X-ray crystal structure of a Ni-methyl species that is a proposed catalytic intermediate in methyl-coenzyme M reductase (MCR), the enzyme that catalyzes the biological formation of methane. The methyl group is situated 2.1 ? proximal of the Ni atom of the MCR coenzyme F(430). A rearrangement of the substrate channel has been posited to bring together substrate species, but Ni(III)-methyl formation alone does not lead to any observable structural changes in the channel.     

Coenzyme F430 from Methanogenic Bacteria: Oxidation of F430 Pentamethyl Ester to the Ni(III) Form

F430M, the pentamethyl ester of coenzyme F430, can be oxidized reversibly by one electron. The oxidation potential has been determined, and the electrolytically prepared oxidation product was characterized by its UV/VIS and ESR spectrum. The strongly anisotropic and nearly axial ESR spectrum is consistent with a S = ½ species with the unpaired-electron spin density predominantly in a d-type orbital of the central nickel ion. The properties of Ni(III)F430M are discussed in the context of two hypothetical mechanisms for the catalytic role of coenzyme F430 in methyl coenzyme M reductase, which catalyses the last step of methane formation in methanogenic bacteria.     

A mechanism from quantum chemical studies for methane formation in methanogenesis

The mechanism for methane formation in methyl-coenzyme M reductase (MCR) has been investigated using the B3LYP hybrid density functional method and chemical models consisting of 107 atoms. The experimental X-ray crystal structure of the enzyme in the inactive MCR(ox1)(-)(silent) state was used to set up the initial model structure. The calculations suggest a mechanism not previously proposed, in which the most remarkable feature is the formation of an essentially free methyl radical at the transition state. The reaction cycle suggested starts from a Michaelis complex with CoB and methyl-CoM coenzymes bound and with a squareplanar coordination of the Ni(I) center in the tetrapyrrole F(430) prosthetic group. In the rate-limiting step the methyl radical is released from methyl-CoM, induced by the attack of Ni(I) on the methyl-CoM thioether sulfur. In this step, the metal center is oxidized from Ni(I) to Ni(II). The resulting methyl radical is rapidly quenched by hydrogen-atom transfer from the CoB thiol group, yielding the methane molecule and the CoB radical. The estimated activation energy is around 20 kcal/mol, which includes a significant contribution from entropy due to the formation of the free methyl. The mechanism implies an inversion of configuration at the reactive carbon. The size of the inversion barrier is used to explain the fact that CF(3)-S-CoM is an inactive substrate. Heterodisulfide CoB-S-S-CoM formation is proposed in the final step in which nickel is reduced back to Ni(I). The suggested mechanism agrees well with experimental observations.     

1-丁烯齐聚反应——Ⅵ.羧酸镍/乙基氯化铝催化体系活性物种的测定

本文利用羧酸镍/乙基氯化铝催化体系催化1-丁烯齐聚反应,用核磁共振和红外光谱技术测定了该体系的催化活性物种。对2-乙基已酸镍/倍半乙基氯化铝体系,在~1H—NMR谱上,于—30.2——33.5 ppm处出现一组信号。对2-乙基己酸镍/乙基二氯化铝体系,在~1H—NMR谱上,分别于-30.2和-34.4ppm处出现信号,在IR谱上,在1955.7cm~(-1)处出现吸收峰。确证具有催化活性的物种为镍氢配合物。     

How Is Methane Formed and Oxidized Reversibly When Catalyzed by Ni‐Containing Methyl‐Coenzyme M Reductase?

Ni‐containing methyl‐coenzyme M reductase (MCR) is capable of catalyzing methane formation and has recently been observed to also be able to catalyze the reverse reaction, the anaerobic oxidation of methane. The forward reaction has been extensively studied theoretically before and was found to consist of two steps. The first step is rate‐limiting and the second step was therefore treated at a lower level. For an accurate treatment of the reverse reaction, both steps have to be studied at the same level. In the present paper, the mechanisms for the reversible formation and oxidation of methane catalyzed by MCR have been investigated using hybrid density functional theory with recent developments, in particular including dispersion effects. An active‐site model was constructed based on the X‐ray crystal structure. The calculations indicate that the MCR reaction is indeed reversible and proceeds via a methyl radical and a Ni‐S(CoM) intermediate with reasonable reaction barriers in both directions. In a competing mechanism, the formation of the crucial Ni‐methyl intermediate, was found to be strongly endergonic by over 20 kcal mol^?1 (including a barrier) with dispersion and entropy effects considered, and thus would not be reachable in a reasonable time under natural conditions.     

Methane formation by reaction of a methyl thioether with a photo-excited nickel thiolate--a process mimicking methanogenesis in archaea

The formation of a sulfuranyl radical intermediate followed by methyl transfer to the nickel(I) center of coenzyme F430 and generation of the disulfide has been proposed as a possible mechanism for the formation of methane catalyzed by methyl coenzyme M reductase in methanogenic archaea. In order to test this hypothesis, a sterically shielded, bifunctional model substrate that contained a methyl thioether and a sulfhydryl functional group, which could form a five-membered cyclic sulfuranyl radical according to the postulated mechanism, was synthesized. The corresponding thiolate reacted with Ni(II) salts to give a diamagnetic, square-planar Ni(II) dithiolate complex, which was characterized by X-ray diffraction. Upon irradiation of this complex with light of lambda > 300 nm, methane and the cyclic disulfide were formed, whereas irradiation of the thiolate in the absence of nickel gave only traces of methane and no cyclic disulfide. The observed products are consistent with the postulated mechanism via a sulfuranyl radical, and the role of light is interpreted as the formation of a Ni(I)/thiyl radical pair upon excitation of a charge-transfer band of the Ni(II) dithiolate. In the presence of a large excess of thiolate, the diamagnetic complex was transformed into a paramagnetic, five- or six-coordinate complex that proved to be more active in the generation of both methane and the cyclic disulfide, than the square-planar diamagnetic dithiolate.     

Nickel oxidation states of F(430) cofactor in methyl-coenzyme M reductase

Magnetic circular dichroism (MCD) spectroscopy and variable-temperature variable-field MCD are used in combination with density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations to characterize the so-called ox1-silent, red1, and ox1 forms of the Ni-containing cofactor F430 in methyl-coenzyme M reductase (MCR). Previous studies concluded that the ox1 state, which is the precursor of the key reactive red1 state of MCR, is a Ni(I) species that derives from one-electron reduction of the Ni(II)-containing ox1-silent state. However, our absorption and MCD data provide compelling evidence that ox1 is actually a Ni(II) species. In support of this proposal, our DFT and TD-DFT calculations indicate that addition of an electron to the ox1-silent state leads to formation of a hydrocorphin anion radical rather than a Ni(I) center. These results and biochemical evidence suggest that ox1 is more oxidized than red1, which prompted us to test a new model for ox1 in which the ox1-silent species is oxidized by one electron to form a thiyl radical derived from coenzyme M that couples antiferromagnetically to the Ni(II) ion. This alternative ox1 model, formally corresponding to a Ni(III)/thiolate resonance form but with predicted S = 1/2 EPR parameters reminiscent of a Ni(I) (3dx2-y2)1 species, rationalizes the requirement for reduction of ox1 to yield the red1 species and the seemingly incongruent EPR and electronic spectra of the ox1 state.     

Structure of an F430 variant from archaea associated with anaerobic oxidation of methane

Microbial mats collected at cold methane seeps in the Black Sea carry out anaerobic oxidation of methane (AOM) to carbon dioxide using sulfate as the electron acceptor. These mats, which predominantly consist of sulfate-reducing bacteria and archaea of the ANME-1 and ANME-2 type, contain large amounts of proteins very similar to methyl-coenzyme M reductase from methanogenic archaea. Mass spectrometry of mat samples revealed the presence of two nickel-containing cofactors in comparable amounts, one with the same mass as coenzyme F430 from methanogens (m/z = 905) and one with a mass that is 46 Da higher (m/z = 951). The two cofactors were isolated and purified, and their constitution and absolute configuration were determined. The cofactor with m/z = 905 was proven to be identical to coenzyme F430 from methanogens. For the m/z = 951 species, high resolution ICP-MS pointed to F430 + CH2S as the molecular formula, and LA-ICP-SF MS finally confirmed the presence of one sulfur atom per nickel. Esterification gave two stereoisomeric pentamethyl esters with m/z = 1021, which could be purified by reverse phase HPLC and were subjected to comprehensive NMR analysis, allowing determination of their constitution and configuration as (17(2)S)-17(2)-methylthio-F430 pentamethyl ester and (17(2)R)-17(2)-methylthio-F430 pentamethyl ester. The corresponding diastereoisomeric pentaacids could also be separated by HPLC and were correlated to the esters via mild hydrolysis of the latter. Equilibration of the pentaacids under acid catalysis showed that the (17(2)S) isomer is the naturally occurring albeit thermodynamically less stable one. The more stable (17(2)R) isomer (80% at equilibrium) is an isolation artifact generated under the acidic conditions necessary for the isolation of the cofactors from the calcium carbonate-encrusted mats.     

Didehydroaspartate Modification in Methyl‐Coenzyme M Reductase Catalyzing Methane Formation

All methanogenic and methanotrophic archaea known to date contain methyl‐coenzyme M reductase (MCR) that catalyzes the reversible reduction of methyl‐coenzyme M to methane. This enzyme contains the nickel porphinoid F₄₃₀ as a prosthetic group and, highly conserved, a thioglycine and four methylated amino acid residues near the active site. We describe herein the presence of a novel post‐translationally modified amino acid, didehydroaspartate, adjacent to the thioglycine as revealed by mass spectrometry and high‐resolution X‐ray crystallography. Upon chemical reduction, the didehydroaspartate residue was converted into aspartate. Didehydroaspartate was found in MCR I and II from Methanothermobacter marburgensis and in MCR of phylogenetically distantly related Methanosarcina barkeri but not in MCR I and II of Methanothermobacter wolfeii, which indicates that didehydroaspartate is dispensable but might have a role in fine‐tuning the active site to increase the catalytic efficiency.