Two sub-states of the red2 state of methyl-coenzyme M reductase revealed by high-field EPR spectroscopy

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 α₂β₂γ₂ subunit structure. Each active site has the nickel porphyrinoid F₄₃₀ as a prosthetic group. In the active state, F₄₃₀ 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. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Probing the reactivity of Ni in the active site of methyl-coenzyme M reductase with substrate analogues

Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH₃-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. It contains the nickel porphyrinoid F₄₃₀ as prosthetic group which has to be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-derived EPR signal MCR-red1. We report here on experiments with methyl-coenzyme M analogues showing how they affect the activity and the MCR-red1 signal of MCR from Methanothermobacter marburgensis. Ethyl-coenzyme M was the only methyl-coenzyme M analogue tested that was used by MCR as a substrate. Ethyl-coenzyme M was reduced to ethane (apparent K _M=20 mM; apparent V _max=0.1 U/mg) with a catalytic efficiency of less than 1% of that of methyl-coenzyme M reduction to methane (apparent K _M=5 mM; apparent V _max=30 U/mg). Propyl-coenzyme M (apparent K _i=2 mM) and allyl-coenzyme M (apparent K _i=0.1 mM) were reversible inhibitors. 2-Bromoethanesulfonate ([I]_{0.5 V}=2 µM), cyano-coenzyme M ([I]_{0.5 V}=0.2 mM), 3-bromopropionate ([I]_{0.5 V}=3 mM), seleno-coenzyme M ([I]_{0.5 V}=6 mM) and trifluoromethyl-coenzyme M ([I]_{0.5 V}=6 mM) irreversibly inhibited the enzyme. In their presence the MRC-red1 signal was quenched, indicating the oxidation of Ni(I) to Ni(II). The rate of oxidation increased over 10-fold in the presence of coenzyme B, indicating that the Ni(I) reactivity was increased in the presence of coenzyme B. Enzyme inactivated in the presence of coenzyme B showed an isotropic signal characteristic of a radical that is spin coupled with one hydrogen nucleus. The coupling was also observed in D₂O. The signal was abolished upon exposure of the enzyme to O₂. 3-Bromopropanesulfonate ([I]_{0.5 V}=0.1 µM), 3-iodopropanesulfonate ([I]_{0.5 V}=1 µM), and 4-bromobutyrate also inactivated MCR. In their presence the EPR signal of MCR-red1 was converted into a Ni-based EPR signal MCR-BPS that resembles in line shape the MCR-ox1 signal. The signal was quenched by O₂. 2-Bromoethanesulfonate and 3-bromopropanesulfonate, which both rapidly reacted with Ni(I) of MRC-red1, did not react with the Ni of MCR-ox1 and MCR-BPS. The Ni-based EPR spectra of both inactive forms were not affected in the presence of high concentrations of these two potent inhibitors.     

The nickel enzyme methyl-coenzyme M reductase from methanogenic archaea: in vitro interconversions among the EPR detectable MCR-red1 and MCR-red2 states

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M and coenzyme B in methanogenic archaea. The enzyme contains tightly bound the nickel porphinoid F430. The nickel enzyme has been shown to be active only when its prosthetic group is in the Ni(I) reduced state. In this state MCR exhibits the nickel-based EPR signal red1. We report here for the MCR from Methanothermobacter marburgensis that the EPR spectrum of the active enzyme changed upon addition or removal of coenzyme M, methyl coenzyme M and/or coenzyme B. In the presence of methyl-coenzyme M the red1 signal showed a more resolved 14N-superhyperfine splitting than in the presence of coenzyme M indicating a possible axial ligation of the substrate to the Ni(I). In the presence of methyl-coenzyme M and coenzyme B the red1 signal was the same as in the presence of methyl-coenzyme M alone. However, in the presence of coenzyme M and coenzyme B a highly rhombic EPR signal, MCR-red2, was induced, which was found to be light sensitive and appeared to be formed at the expense of the MCR-red1 signal. Upon addition of methyl-coenzyme M, the red2 signal disappeared and the red1 signal increased again. The red2 signal of MCR with 61Ni-labeled cofactor was significantly broadened indicating that the signal is nickel or nickel-ligand based.     

Coordination and binding geometry of methyl-coenzyme M in the red1m state of methyl-coenzyme M reductase

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 MCR_red1 (red for reduced Ni) have been identified. Here, we report a detailed characterization of MCR_red1m (“m” for methyl-coenzyme M), which is the complex of MCR_red1a (“a” for absence of substrate) with CH₃-S-CoM. Using continuous-wave and pulse electron paramagnetic resonance spectroscopy in combination with selective isotope labeling (¹³C and ²H) 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 substrate, 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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme M reductase upon substrate binding

Methyl-coenzyme M reductase (MCR) catalyzes the final reaction of the energy conserving pathway of methanogenic archaea in which methylcoenzyme M and coenzyme B are converted to methane and the heterodisulfide CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisulfide CoM-S-S-CoB (MCR-silent) were now refined at 1.16 A and 1.8 A resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F430, of the active site residues and of the modified amino acid residues. Moreover, the observation of 18 Mg2+ and 9 Na+ ions at the protein surface of the 300 kDa enzyme specifies typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased flexibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced specific conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.     

Characterization of the MCRred2 form of methyl-coenzyme M reductase: a pulse EPR and ENDOR study

Methyl-coenzyme M reductase (MCR), which catalyses the reduction of methyl-coenzyme M (CH(3)-S-CoM) with coenzyme B (H-S-CoB) to CH(4) and CoM-S-S-CoB, contains the nickel porphinoid F430 as prosthetic group. The active enzyme exhibits the Ni(I)-derived axial EPR signal MCR(red1) both in the absence and presence of the substrates. When the enzyme is competitively inhibited by coenzyme M (HS-CoM) the MCR(red1) signal is partially converted into the rhombic EPR signal MCR(red2). To obtain deeper insight into the geometric and electronic structure of the red2 form, pulse EPR and ENDOR spectroscopy at X- and Q-band microwave frequencies was used. Hyperfine interactions of the four pyrrole nitrogens were determined from ENDOR and HYSCORE data, which revealed two sets of nitrogens with hyperfine couplings differing by about a factor of two. In addition, ENDOR data enabled observation of two nearly isotropic (1)H hyperfine interactions. Both the nitrogen and proton data indicate that the substrate analogue coenzyme M is axially coordinated to Ni(I) in the MCR(red2) state.     

Substrate-analogue-induced changes in the nickel-EPR spectrum of active methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum.

Methyl-coenzyme-M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M [2-(methylthio)ethanesulfonate] and 7-mercaptoheptanoylthreonine phosphate in methanogenic archaea. The enzyme contains the nickel porphinoid coenzyme F430 as a prosthetic group. In the active, reduced (red) state, the enzyme displays two characteristic EPR signals, MCR-red1 and MCR-red2, probably derived from Ni(I). In the presence of the substrate methyl-coenzyme M, the rhombic MCR-red2 signal is quantitatively converted to the axial MCR-red1 signal. We report here on the effects of inhibitory substrate analogues on the EPR spectrum of the enzyme. 3-Bromopropanesulfonate (BrPrSO3), which is the most potent inhibitor of MCR known to date (apparent Ki = 0.05 microM), converted the EPR signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal designated MCR-BrPrSO3. 3-Fluoropropanesulfonate (apparent Ki < 50 microM) and 3-iodopropanesulfonate (apparent Ki < 1 microM) induced a signal identical to that induced by BrPrSO3 without affecting the line shape, despite the fact that the fluorine, bromine and iodine isotopes employed have nuclear spins of I = 1/2, I = 3/2 and I = 5/2, respectively. This finding suggests that MCR-BrPrSO3 is not the result of a close halogen-Ni(I) interaction. 7-Bromoheptanoylthreonine phosphate (BrHpoThrP) (apparent Ki = 5 microM), which is an inhibitory substrate analogue of 7-mercaptoheptanoylthreonine phosphate, converted the signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal, MCR-BrHpoThrP, similar but not identical to MCR-BrPrSO3. The results indicate that inhibition of MCR by the halogenated substrate analogues investigated above is not via oxidation of Ni(I)F430. The different MCR EPR signals are assigned to different enzyme/substrate and enzyme/inhibitor complexes.     

Spectroscopic investigation of the nickel-containing porphinoid cofactor F<Subscript>430</Subscript>. Comparison of the free cofactor in the +1, +2 and +3 oxidation states with the cofactor bound to methyl-coenzyme M reductase in the silent,red and ox forms

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. It contains the nickel porphinoid F₄₃₀, a prosthetic group that has been proposed to be directly involved in the catalytic cycle by the direct binding and subsequent reduction of the substrate methyl-coenzyme M. The active enzyme (MCRred1) can be generated in vivo and in vitro by reduction from MCRox1, which is an inactive form of the enzyme. Both the MCRred1 and MCRox1 forms have been proposed to contain F₄₃₀ in the Ni(I) oxidation state on the basis of EPR and ENDOR data. In order to further address the oxidation state of the Ni center in F₄₃₀, variable-temperature, variable-field magnetic circular dichroism (VTVH MCD), coupled with parallel absorption and EPR studies, have been used to compare the electronic and magnetic properties of MCRred1, MCRox1, and various EPR silent forms of MCR, with those of the isolated penta-methylated cofactor (F₄₃₀M) in the +1, +2 and +3 oxidation states. The results confirm Ni(I) assignments for MCRred1 and MCRred2 forms of MCR and reveal charge transfer transitions involving the Ni d orbitals and the macrocycle orbitals that are unique to Ni(I) forms of F₄₃₀. Ligand field transitions associated with S=1 Ni(II) centers are assigned in the near-IR MCD spectra of MCRox1-silent and MCR-silent, and the splitting in the lowest energy d–d transition is shown to correlate qualitatively with assessments of the zero-field splitting parameters determined by analysis of VTVH MCD saturation magnetization data. The MCD studies also support rationalization of MCRox1 as a tetragonally compressed Ni(III) center with an axial thiolate ligand or a coupled Ni(II)-thiyl radical species, with the reality probably lying between these two extremes. The reinterpretation of MCRox1 as a formal Ni(III) species rather than an Ni(I) species obviates the need to invoke a two-electron reduction of the F₄₃₀ macrocyclic ligand on reductive activation of MCRox1 to yield MCRred1.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0549-9Abbreviations F₄₃₀ cofactor 430 - F₄₃₀M penta-methylated form of cofactor 430 - Ni(I)F₄₃₀M F₄₃₀M with the nickel atom in the +1 oxidation state - Ni(II)F₄₃₀M F₄₃₀M with the nickel atom in the +2 oxidation state - Ni(III)F₄₃₀M F₄₃₀M with the nickel atom in the +3 oxidation state - MCR methyl-coenzyme M reductase - MCRox1 MCR exhibiting the MCR-ox1 EPR signal - MCRox1-silent EPR silent form of MCR obtained from the MCRox1 form - MCRred1 MCR exhibiting the EPR signals red1c and/or red1m - MCRred1c MCRred1 in the presence of coenzyme M - MCRred1m MCRred1 in the presence of methyl-coenzyme M - MCRred2 MCR exhibiting both the red1 and red2 EPR signals - MCRred1-silent EPR silent form of MCR obtained from the MCRred1 form - MCRsilent EPR silent form of MCR     

Spectroscopic and kinetic studies of the reaction of bromopropanesulfonate with methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis in which coenzyme B and methyl-coenzyme M are converted to methane and the heterodisulfide, CoMS-SCoB. MCR also appears to initiate anaerobic methane oxidation (reverse methanogenesis). At the active site of MCR is coenzyme F430, a nickel tetrapyrrole. This paper describes the reaction of the active MCR(red1) state with the potent inhibitor, 3-bromopropanesulfonate (BPS; I50 = 50 nM) by UV-visible and EPR spectroscopy and by steady-state and rapid kinetics. BPS was shown to be an alternative substrate of MCR in an ionic reaction that is coenzyme B-independent and leads to debromination of BPS and formation of a distinct state ("MCR(PS)") with an EPR signal that was assigned to a Ni(III)-propylsulfonate species (Hinderberger, D., Piskorski, R. P., Goenrich, M., Thauer, R. K., Schweiger, A., Harmer, J., and Jaun, B. (2006) Angew. Chem. Int. Ed. Engl. 45, 3602-3607). A similar EPR signal was generated by reacting MCR(red1) with several halogenated sulfonate and carboxylate substrates. In rapid chemical quench experiments, the propylsulfonate ligand was identified by NMR spectroscopy and high performance liquid chromatography as propanesulfonic acid after protonolysis of the MCR(PS) complex. Propanesulfonate formation was also observed in steady-state reactions in the presence of Ti(III) citrate. Reaction of the alkylnickel intermediate with thiols regenerates the active MCR(red1) state and eliminates the propylsulfonate group, presumably as the thioether. MCR(PS) is catalytically competent in both the generation of propanesulfonate and reformation of MCR(red1). These results provide evidence for the intermediacy of an alkylnickel species in the final step in anaerobic methane oxidation and in the initial step of methanogenesis.     

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

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 F(430), which is active in the Ni(I) state (MCR(red1)). 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 accumulate to detectible amounts during the reaction between methyl-SCoM and CoBSH. Here, we describe the reaction of methyl-SCoM with a substrate analogue (CoB(6)SH) in which the seven-carbon heptanoyl moiety of CoBSH has been replaced with a hexanoyl group. When MCR(red1) is reacted with methyl-SCoM and CoB(6)SH, methanogenesis occurs 1000-fold more slowly than with CoBSH. By transient kinetic methods, we observe decay of the active Ni(I) state coupled to formation and subsequent decay of alkyl-Ni(III) and organic radical intermediates at catalytically competent rates. The kinetic data also revealed substrate-triggered conformational changes in active Ni(I)-MCR(red1). Electron paramagnetic resonance (EPR) studies coupled with isotope labeling experiments demonstrate that the radical intermediate is not tyrosine-based. These observations provide support for a mechanism for MCR that involves methyl-Ni(III) and an organic radical as catalytic intermediates. Thus, the present study provides important mechanistic insights into the mechanism of this key enzyme that is central to biological methane formation.     

The nickel enzyme methyl-coenzyme M reductase from methanogenic archaea: In vitro induction of the nickel-based MCR-ox EPR signals from MCR-red2

Methyl-coenzyme M reductase (MCR) is a nickel enzyme catalyzing the formation of methane from methyl-coenzyme M and coenzyme B in all methanogenic archaea. The active purified enzyme exhibits the axial EPR signal MCR-red1 and in the presence of coenzyme M and coenzyme B the rhombic signal MCR-red2, both derived from Ni(I). Two other EPR-detectable states of the enzyme have been observed in vivo and in vitro designated MCR-ox1 and MCR-ox2 which have quite different nickel EPR signals and which are inactive. Until now the MCR-ox1 and MCR-ox2 states could only be induced in vivo. We report here that in vitro the MCR-red2 state is converted into the MCR-ox1 state by the addition of polysulfide and into a light-sensitive MCR-ox2 state by the addition of sulfite. In the presence of O(2) the MCR-red2 state was converted into a novel third state designated MCR-ox3 and exhibiting two EPR signals similar but not identical to MCR-ox1 and MCR-ox2. The formation of the MCR-ox states was dependent on the presence of coenzyme B. Investigations with the coenzyme B analogues S-methyl-coenzyme B and desulfa-methyl-coenzyme B indicate that for the induction of the MCR-ox states the thiol group of coenzyme B is probably not of importance. The results were obtained with purified active methyl-coenzyme M reductase isoenzyme I from Methanothermobacter marburgensis. They are discussed with respect to the nickel oxidation states in MCR-ox1, MCR-ox2 and MCR-ox3 and to a possible presence of a second redox active group in the active site. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00775-001-0325-z.     

Coordination and geometry of the nickel atom in active methyl-coenzyme M reductase from Methanothermobacter marburgensis as detected by X-ray absorption spectroscopy

Methyl-coenzyme M reductase (MCR) catalyzes the reduction of methyl-coenzyme M (CH(3)-S-CoM) to methane. The enzyme contains as a prosthetic group the nickel porphinoid F(430) which in the active enzyme is in the EPR-detectable Ni(I) oxidation state. Crystal structures of several inactive Ni(II) forms of the enzyme but not of the active Ni(I) form have been reported. To obtain structural information on the active enzyme-substrate complex we have now acquired X-ray absorption spectra of active MCR in the presence of either CH(3)-S-CoM or the substrate analog coenzyme M (HS-CoM). For both MCR complexes the results are indicative of the presence of a five-coordinate Ni(I), the five ligands assigned as four nitrogen ligands from F(430) and one oxygen ligand. Analysis of the spectra did not require the presence of a sulfur ligand indicating that CH(3)-S-CoM and HS-CoM were not coordinated via their sulfur atom to nickel in detectable amounts. As a control, X-ray absorption spectra were evaluated of three enzymatically inactive MCR forms, MCR-silent, MCR-ox1-silent and MCR-ox1, in which the nickel is known to be six-coordinate. Comparison of the edge position of the X-ray absorption spectra revealed that the Ni(I) in the active enzyme is more reduced than the Ni in the two EPR-silent Ni(II) states. Surprisingly, the edge position of the EPR-active MCR-ox1 state was found to be the same as that of the two silent states indicating similar electron density on the nickel.     

Elucidating the Process of Activation of Methyl-Coenzyme M Reductase

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 MCR_silent-Ni(II) form to the active MCR_red1-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 MCR_red1, 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.     

Characterization of alkyl-nickel adducts generated by reaction of methyl-coenzyme m reductase with brominated acids

Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the final step in the biological synthesis of methane. Using coenzyme B (CoBSH) as the two-electron donor, MCR reduces methyl-coenzyme M (methyl-SCoM) to methane and the mixed disulfide, CoB-S-S-CoM. MCR contains coenzyme F430, an essential redox-active nickel tetrahydrocorphin, at its active site. The active form of MCR (MCRred1) contains Ni(I)-F430. When 3-bromopropane sulfonate (BPS) is incubated with MCRred1, an alkyl-Ni(III) species is formed that elicits the MCRPS EPR signal. Here we used EPR and UV-visible spectroscopy and transient kinetics to study the reaction between MCR from Methanothermobacter marburgensis and a series of brominated carboxylic acids, with carbon chain lengths of 4-16. All of these compounds give rise to an alkyl-Ni intermediate with an EPR signal similar to that of the MCRPS species. Reaction of the alkyl-Ni(III) adduct, formed from brominated acids with eight or fewer total carbons, with HSCoM as nucleophile at pH 10.0 results in the formation of a thioether coupled to regeneration of the active MCRred1 state. When reacted with 4-bromobutyrate, MCRred1 forms the alkyl-Ni(III) MCRXA state and then, surprisingly, undergoes "self-reactivation" to regenerate the Ni(I) MCRred1 state and a bromocarboxy ester. The results demonstrate an unexpected reactivity and flexibility of the MCR active site in accommodating a broad range of substrates, which act as molecular rulers for the substrate channel in MCR.     

Characterization of the thioether product formed from the thiolytic cleavage of the alkyl-nickel bond in methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in methanogenesis by using N-7-mercaptoheptanolyl-threonine phosphate (CoBSH) as the two-electron donor to reduce 2-(methylthiol)ethane sulfonate (methyl-SCoM) to methane, and producing the heterodisulfide, CoBS-SCoM. The active site of MCR includes a noncovalently bound Ni tetrapyrrolic cofactor called coenzyme F430, which is in the Ni(I) state in the active enzyme (MCRred1). Bromopropanesulfonate (BPS) is a potent inhibitor and reversible redox inactivator that reacts with MCRred1 to form an EPR-active state called MCRPS, which is an alkyl-nickel species. When MCRPS is treated with free thiol containing compounds, the enzyme is reconverted to the active MCRred1 state. In this paper, we demonstrate that the reactivation of MCRPS to MCRred1 by thiols involves formation of a thioether product. MCRPS also can be converted to active MCRred1 by treatment with sodium borohydride. Reactivation is highest with the smallest free thiol HS-. Interestingly, MCRPS can also be reductively activated with analogues of CoBSH such as CoB8SH and CoB9SH, but not CoBSH itself. Unambiguous demonstration of the formation of a methylthioether product from thiolysis of an alkyl-Ni species provides support for a methyl-Ni intermediate in the MCR-catalyzed last step in methanogenesis and the first proposed step in anaerobic methane oxidation.     

A F420-dependent NADP reductase in the extremely thermophilic sulfate-reducing Archaeoglobus fulgidus

Archaeoglobus fulgidus, a sulfate-reducing Archaeon with a growth temperature optimum of 83°C, uses the 5-deazaflavin coenzyme F₄₂₀ rather than pyridine nucleotides in catabolic redox processes. The organism does, however, require reduced pyridine nuclcotides for biosynthetic purposes. We describe here that the Archaeon contains a coenzyme F₄₂₀-dependent NADP reductase which links anabolism to catabolism. The highly thermostable enzyme was purfied 3600-fold by affinity chromatography to apparent homogeneity in a 60% yield. The native enzyme with an apparent molecular mass of 55 kDa was composed of only one type of subunit of apparent molecular mass of 28 kDa. Spectroscopic analysis of the enzyme did not reveal the presence of any chromophoric prosthetic group. The purified enzyme catalyzed the reversible reduction of NADP (apparent K _M 40 M) with reduced F₄₂₀ (apparent K _M 20M) with a specific activity of 660 U/mg (apparent V _max) at pH 8.0 (pH optimum) and 80°C (temperature optimum). It was specific for both coenzyme F₄₂₀ and NADP. Sterochemical investigations showed that the F₄₂₀-dependent NADP reductase was Si face specific with respect to C5 of F₄₂₀ and Si face specific with respect to C4 of NADP.Abbreviations F₄₂₀ coenzyme F₄₂₀ - F₄₂₀H₂ 1,5-dihydrocoenzyme F₄₂₀ - H₄MPT tetrahydromethanopterin - CH=H₄MPT N⁵, N¹⁰-methylenetetrahydromethanopterin - MFR methanofuran - HPLC high performance liquid chromatography - methylene-H₄MPT dehydrogenase N⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase - 1 U = 1 mol/min     

Methyl-coenzyme M reductase and other enzymes involved in methanogenesis from CO2 and H2 in the extreme thermophile Methanopyrus kandleri

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H₂ and CO₂ and that shows no close phylogenetic relationship to any methanogen known so far. Methyl-coenzyme M reductase, the enzyme catalyzing the methane forming step in the energy metabolism of methanogens, was purified from this hyperthermophile. The yellow protein with an absorption maximum at 425 nm was found to be similar to the methyl-coenzyme M reductase from other methanogenic bacteria in that it was composed each of two -, - and -subunits and that it contained the nickel porphinoid coenzyme F₄₃₀ as prosthetic group. The purified reductase was inactive. The N-terminal amino acid sequence of the -subunit was determined. A comparison with the N-terminal sequences of the -subunit of methyl-coenzyme M reductases from other methanogenic bacteria revealed a high degree of similarity.Besides methyl-coenzyme M reductase cell extracts of M. kandleri were shown to contain the following enzyme activities involved in methanogenesis from CO₂ (apparent V_max at 65°C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran: tetrahydromethanopterin formyltransferase, 13 U/mg; N ⁵,N¹⁰-methenyltetrahydromethanopterin cyclohydrolase, 14 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin dehydrogenase (H₂-forming), 33 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin reductase (coenzyme F₄₂₀ dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F₄₂₀-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg. Apparent K_m values for these enzymes and the effect of salts on their activities were determined.The coenzyme F₄₂₀ present in M. kandleri was identified as coenzyme F₄₂₀-2 with 2 -glutamyl residues.Abbreviations H–S-CoM coenzyme M - CH₃–S-CoM methylcoenzyme M - H–S-HTP 7-mercaptoheptanoylthreonine phosphate - MFR methanofuran - CHO-MFR formyl-MFR - H₄MPT tetrahydromethanopterin - CHO–H₄MPT N ⁵-formyl-H₄MPT - CH=H₄MPT⁺ N ⁵,N¹⁰-methenyl-H₄MPT - CH₂=H₄MPT N ⁵,N¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - 1 U= 1 mol/min     

Comparison of three methyl-coenzyme M reductases from phylogenetically distant organisms: unusual amino acid modification, conservation and adaptation

The nickel enzyme methyl-coenzyme M reductase (MCR) catalyzes the terminal step of methane formation in the energy metabolism of all methanogenic archaea. In this reaction methyl-coenzyme M and coenzyme B are converted to methane and the heterodisulfide of coenzyme M and coenzyme B. The crystal structures of methyl-coenzyme M reductase from Methanosarcina barkeri (growth temperature optimum, 37 degrees C) and Methanopyrus kandleri (growth temperature optimum, 98 degrees C) were determined and compared with the known structure of MCR from Methanobacterium thermoautotrophicum (growth temperature optimum, 65 degrees C). The active sites of MCR from M. barkeri and M. kandleri were almost identical to that of M. thermoautotrophicum and predominantly occupied by coenzyme M and coenzyme B. The electron density at 1.6 A resolution of the M. barkeri enzyme revealed that four of the five modified amino acid residues of MCR from M. thermoautotrophicum, namely a thiopeptide, an S-methylcysteine, a 1-N-methylhistidine and a 5-methylarginine were also present. Analysis of the environment of the unusual amino acid residues near the active site indicates that some of the modifications may be required for the enzyme to be catalytically effective. In M. thermoautotrophicum and M. kandleri high temperature adaptation is coupled with increasing intracellular concentrations of lyotropic salts. This was reflected in a higher fraction of glutamate residues at the protein surface of the thermophilic enzymes adapted to high intracellular salt concentrations.     

Comparative analysis of genes encoding methyl coenzyme M reductase in methanogenic bacteria

Summary The sequence of the gene cluster encoding the methyl coenzyme M reductase (MCR) in Methanococcus voltae was determined. It contains five open reading frames (ORF), three of which encode the known enzyme subunits. Putative ribosome binding sites were found in front of all ORFs. They differ in their degrees of complementarity to the 3 end of the 16 S rRNA, which is discussed in terms of different translation efficiencies of the respective genes. The codon usage bias is different in the subunit encoding genes compared with the two other ORFs in the cluster and two other known genes of Mc. voltae. This is interpreted in terms of increased translational accuracy of the highly expressed MCR subunit genes. The derived polypeptide sequences encoded by the five ORFs of the MCR cluster were compared to those of the respective genes in Methanobacterium thermoautotrophicum Marburg and Methanosarcina barkeri. Conserved regions were detected in the enzyme subunits, which are candidates for factor binding domains. Conserved hydrophobic sequences found in the and subunits are discussed with respect to the membrane association of the enzyme.     

Spectroscopic and computational studies of reduction of the metal versus the tetrapyrrole ring of coenzyme F430 from methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) catalyzes the final step in methane biosynthesis by methanogenic archaea and contains a redox-active nickel tetrahydrocorphin, coenzyme F430, at its active site. Spectroscopic and computational methods have been used to study a novel form of the coenzyme, called F330, which is obtained by reducing F430 with sodium borohydride (NaBH4). F330 exhibits a prominent absorption peak at 330 nm, which is blue shifted by 100 nm relative to F430. Mass spectrometric studies demonstrate that the tetrapyrrole ring in F330 has undergone reduction, on the basis of the incorporation of protium (or deuterium), upon treatment of F430 with NaBH4 (or NaBD4). One- and two-dimensional NMR studies show that the site of reduction is the exocyclic ketone group of the tetrahydrocorphin. Resonance Raman studies indicate that elimination of this pi-bond increases the overall pi-bond order in the conjugative framework. X-ray absorption, magnetic circular dichroism, and computational results show that F330 contains low-spin Ni(II). Thus, conversion of F430 to F330 reduces the hydrocorphin ring but not the metal. Conversely, reduction of F430 with Ti(III) citrate to generate F380 (corresponding to the active MCR(red1) state) reduces the Ni(II) to Ni(I) but does not reduce the tetrapyrrole ring system, which is consistent with other studies [Piskorski, R., and Jaun, B. (2003) J. Am. Chem. Soc. 125, 13120-13125; Craft, J. L., et al. (2004) J. Biol. Inorg. Chem. 9, 77-89]. The distinct origins of the absorption band shifts associated with the formation of F330 and F380 are discussed within the framework of our computational results. These studies on the nature of the product(s) of reduction of F430 are of interest in the context of the mechanism of methane formation by MCR and in relation to the chemistry of hydroporphinoid systems in general. The spectroscopic and time-dependent DFT calculations add important insight into the electronic structure of the nickel hydrocorphinate in its Ni(II) and Ni(I) valence states.