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.     

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.     

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.     

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.     

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.     

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.     

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.     

A nickel hydride complex in the active site of methyl-coenzyme m reductase: implications for the catalytic cycle

Methanogenic archaea utilize a specific pathway in their metabolism, converting C1 substrates (i.e., CO2) or acetate to methane and thereby providing energy for the cell. Methyl-coenzyme M reductase (MCR) catalyzes the key step in the process, namely methyl-coenzyme M (CH3-S-CoM) plus coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. The active site of MCR contains the nickel porphinoid F430. We report here on the coordinated ligands of the two paramagnetic MCR red2 states, induced when HS-CoM (a reversible competitive inhibitor) and the second substrate HS-CoB or its analogue CH3-S-CoB are added to the enzyme in the active MCR red1 state (Ni(I)F430). Continuous wave and pulse EPR spectroscopy are used to show that the MCR red2a state exhibits a very large proton hyperfine interaction with principal values A((1)H) = [-43,-42,-5] MHz and thus represents formally a Ni(III)F430 hydride complex formed by oxidative addition to Ni(I). In view of the known ability of nickel hydrides to activate methane, and the growing body of evidence for the involvement of MCR in "reverse" methanogenesis (anaerobic oxidation of methane), we believe that the nickel hydride complex reported here could play a key role in helping to understand both the mechanism of "reverse" and "forward" methanogenesis.     

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.     

Structural and functional analogue of the active site of polysulfide reductase from Wolinella succinogenes

Synthesis of [PPh4]2[Mo(SPh)2(S2C2(CN)2)2] (2) from [PPh4]2[MoO(S2C2(CN)2)2] (1) has been achieved to mimic the postulated [Mo(S)6] core of polysulfide reductase with two thiolates and two bis(ene-dithiolate) ligands. Compound 2 reacts with polysulfide to yield H2S, modeling the function of polysulfide reductase. The facile conversion of 2 back to 1 in moist solvent suggests that the interconversion of the [MoIV = O] and [MoIV - X] (X = O-Ser, S-Cys, Se-Cys) moieties might occur in the DMSO reductase class of enzymes under appropriate hydrophobic/hydrophilic conditions.     

Structural analysis of reactive dye species retained by the basic alumina surface

The alumina-dye composites were prepared by treating the basic alumina with the water solutions of Reactive Red 120 (RR 120) and Reactive Blue 15 (RB 15) dyes. The bands of low intensities in the 1400–1600 cm⁻¹ region and at 783 cm⁻¹ in the IR spectra of these composites point out that the dye species is bound weakly to the surface. In the case of mechanochemical adsorption of dye molecules, the asymmetric and symmetric S(=O)₂ and the S-O-C stretching bands together with the vibrations of aromatic ring revealed that dye types under dry conditions interacted effectively with alumina surface. After the heating of the alumina dye complexes in the temperature range 150–350°C, the intensities of the IR and XRD peaks for adsorbed types decreased. The endothermic peaks over 200°C and the bigger total mass losses for the alumina-dye composites can be ascribed to the decomposition of dye species retained by the alumina surface. The mass losses on TG curves of the alumina-dye complexes up to ∼800°C exhibit the removal of black residues occurred by decomposition of first adsorbed products. The thermal analysis data also point out that the water molecules bonded strongly to the alumina surface and dye types compete to accommodate at the surface active sites.     

Structural analysis and immunomodulatory activity of a homopolysaccharide isolated from Parabacteroides distasonis

A homopolysaccharide fraction (PEP-1A) with desired immunomodulatory activity was isolated from culture broth of Parabacteroides distasonis. Structural characterization uncovered that PEP-1A had a molecular weight of 3.40 × 10⁶ Da and possessed mannose merely. Comprehensive analysis of FT-IR, GC–MS and 1D/2D NMR confirmed that the structure of PEP-1A was expected as follows:

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Finally, the activity assays on RAW 264.7 macrophages showed that PEP-1A could promote proliferation, increase the production of reactive oxygen species (ROS), enhance the phagocytosis, and promote the secretion of nitric oxide (NO) and inflammatory cytokines including IL-1β, IL-6 and TNF-α. Furthermore, PEP-1A could give play to immunoregulation effect through NF-κB, MAPK and Akt signaling pathways. Collectively, the study of PEP-1A offers a molecular underpinning for the future application of PEP-1A as a potential immunostimulant.     

Structural analysis of the gum polysaccharide from anacardium occidentale

The gum exudate from Anacardium occidentale contains galactose (61 %), arabinose (14 %), rhamnose (7 %), glucose (8 %) and glucuronic acid (5 %) in addition to small amounts (<2 %) of each of mannose, xylose and 4-0 methylglucuronic acid. Contrary to earlier findings, the main aldobiuronic acid present is 6-O-(β-D-glucopyranosyluronic acid)-D-galactose; smaller amounts of the 4-O-methyl analogue are also present. Mild acid hydrolysis showed only two galactobioses, 3-O-β-D-galactopyranosyl-D-galactose (major

component) and 6-O-β-D-galactopyranosyl-D-galactose (minor component). Degraded gum A, prepared by controlled acid hydrolysis, contained galactose, glucose, and uronic acid. A Smith-degradation of degraded gum A gave degraded gum B, which contained only galactose. Sequential Smith-degradations of Anacardium occidentale gum, and methylation analyses of the gum and of its degradation products indicated a highly-branched galactan framework consisting of chains of β-(1–3)-linked D-galactose residues branched and interspersed with β-(1–6) linkages. Arabinose is present as end-groups or in short (1–2)-linked chains up to five units long. Glucose, rhamnose, mannose xylose, and uronic acid are all present as end-groups.     

Mercury analysis: a special example of species analysis

Summary Mercury species analysis requires the determination of numerous different compounds with very different behaviour regarding environment and toxicity. For these differing species several new and more sensitive analytical methods have been developed and tested. Mercury species cannot be detected directly in most cases, and are normally derivatized by different agents and determined by different detector systems. In a new HPLC method with photometric detection more than 10 different organomercurials could be analysed as their thio-ethanol complexes. By alkylation of mercury compounds they can be separated by GC and analysed with an AAS or AFS as detector. Volatile organic species of Hg can be separated on Carbotrap^®, where Hg⁰ is not adsorbed, and analysed thereafter. With atomic fluorescence the detection limit for Hg⁰ measurement is improved significantly when compared with the widely used atomic absorption method. Results obtained with this new method are shown and discussed. Future developments for species analysis are outlined; not only to analyse the covalent metal — carbon/oxygen compounds — primary species-analysis — but also compounds with ionic or complex bonding: secondary species-analysis. With these developments, prediction of species behaviour in the environment, including toxicity assessment and decontamination proposals, should be made possible.     

Structural information from detailed analysis of infrared absorption bands

The OH-stretching band of more than 500 alcohols and phenols have been measured in the apolar solvents CC1₄ and CS₂ under standard conditions and the band parameters have been determined. Based on these data a system is being developed which provides the molecular structure in the surroundings of the hydroxyl group. The scheme of the system is outlined and discussed.     

Structural analysis of a melaminium polyphosphate from X-ray powder diffraction and solid-state NMR data

The crystal structure of the environmentally friendly flame retardant melaminium polyphosphate (MPoly) (2,4,6-triamino-1,3,5-triazinium x PO(3))(n)was determined by a direct-space global optimization technique from X-ray powder diffraction data. Solid-state NMR was used to corroborate the proposed hydrogen-bonding model and to determine the average degree of polymerization (n > 100). An analysis of the crystal structure of MPoly reveals aspects of molecular geometry and packing that are characteristic for melamine-containing compounds and polyphosphate salts. A comparison of MPoly with the crystal structures of its precursors melaminium orthophosphate (MP) and melaminium dihydrogenpyrophosphate (MPy) provides insight in the mechanism of the endothermic dehydration processes that takes place in the reaction path MP --> MPy --> MPoly. Solid-state NMR characterization of various samples of the same batch showed inhomogeneities in the MPoly composition. Various quantities of orthophosphates were found, which cannot be assigned to be MP.     

Protein flexibility and species specificity in structure-based drug discovery: dihydrofolate reductase as a test system

In structure-based drug discovery, researchers would like to identify all possible scaffolds for a given target. However, techniques that push the boundaries of chemical space could lead to many false positives or inhibitors that lack specificity for the target. Is it possible to broadly identify the appropriate chemical space for the inhibitors and yet maintain target specificity? To address this question, we have turned to dihydrofolate reductase (DHFR), a well-studied metabolic enzyme of pharmacological relevance. We have extended our multiple protein structure (MPS) method for receptor-based pharmacophore models to use multiple X-ray crystallographic structures. Models were created for DHFR from human and Pneumocystis carinii. These models incorporate a fair degree of protein flexibility and are highly selective for known DHFR inhibitors over drug-like non-inhibitors. Despite sharing a highly conserved active site, the pharmacophore models reflect subtle differences between the human and P. carinii forms, which identify species-specific, high-affinity inhibitors. We also use structures of DHFR from Candida albicans as a counter example. The available crystal structures show little flexibility, and the resulting models give poorer performance in identifying species-specific inhibitors. Therapeutic success for this system may depend on achieving species specificity between the related human host and these key fungal targets. The MPS technique is a promising advance for structure-based drug discovery for DHFR and other proteins of biomedical interest.