A New Mechanism for Methane Production from ... - ACS Publications

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J. Phys. Chem. B 2008, 112, 2466-2482

A New Mechanism for Methane Production from Methyl-Coenzyme M Reductase As Derived from Density Functional Calculations Evert C. Duin and Michael L. McKee* Department of Chemistry and Biochemistry, Auburn UniVersity, Auburn, Alabama 36849 ReceiVed: October 9, 2007; In Final Form: December 2, 2007

We propose a new DFT-based mechanism for methane production using the full F430 cofactor of MCR (methylcoenzyme M reductase) along with a coordinated OdCH2CH2C(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 SCH2CH2SO32- 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.

Introduction Methanogenesis, the production of CH4 by archaea, has an important influence on life on this planet. First of all, methanogenesis is the last step in the anaerobic breakdown of biopolymers and takes place in many anaerobic microbial habitats such as swamps, rice paddies, freshwater sediments, and the intestinal tract of animals and insects. Additionally, CH4 is a potent greenhouse gas. Although part of the CH4 produced by methanogens is reoxidized by other organisms, more and more CH4 escapes to the atmosphere, mainly due to increasing areas for rice production and the increase in amounts of livestock.1 Methanogens utilize different metabolic pathways for the conversion of the different substrates into methane. These substrates include CO2, formate, acetate, methanol, and methylamines.2 All pathways, however, use the same last step, the conversion of methyl-coenzyme M into CH4 by the enzyme methyl-coenzyme M reductase (MCR), making MCR the central protein in methanogenesis. Although the enzyme was first isolated in 1981,3 only in recent years have we seen the development of methods to purify highly active MCR. This opened up the way to properly study the different states of MCR using biophysical methods. Several recent reviews give in-depth overviews of these investigations.2,4-14 Although there is a good understanding of the properties of the different states that can be detected in MCR, there is limited information available on the actual reaction mechanism. When methyl-coenzyme M (CH3SCoM-, methylthioethane sulfonate) is converted to CH4, coenzyme B (HSCoB, thioheptanoyl threonine phosphate) forms a mixed disulfide with coenzyme M (CoBS-SCoM) in a reaction that is spontaneous by 7.2 kcal/mol (Figure 1).2 The active site of MCR contains coenzyme F430 (Figure 2), which consists of a nickel atom coordinated to a corphin ring (Ni-porphinoid) which is embedded at the end of a channel. The corphin ring, the most extensively reduced tetrapyrrole found in Nature, is quite

flexible, which may be important in the methane-production mechanism.14-19 Due to the very narrow channel, CH3SCoMmust enter with the sulfonate group leading.20,21 After CH3SCoMhas coordinated, the channel is filled by HSCoB where its thiol group approaches the nickel no closer than about 8.0 Å due to strong interactions between the charged groups of HSCoB and MCR at the channel entrance.22 A variety of MCR species have been characterized. In MCRox1-silent, which was solved by X-ray diffraction22 (Figure 3), the Ni(II) center is coordinated to the four nitrogens of the corphin ring, the thiol sulfur of the SCH2CH2SO32- ligand, and an oxygen atom from the Gln147 residue. The HSCoB cofactor is shown in Figure 3 occupying the channel entrance. The X-ray structure of MCRsilent, another form of MCR, exhibits the heterodisulfide (CoMS-SCoB) cofactor coordinated to the Ni(II) center through the sulfonate oxygen.22 The forms that were crystallized are inactive forms with the nickel in the 2+ oxidation state. Several other forms can be detected both in vitro and in whole cells that seem to be relevant for the functioning of this enzyme (Figure 4). First there is the MCRred1 form that is the only actual active form of the enzyme. It can be made to accumulate in the cell by using a more reducing environment (100% H2 vs 80% H2/20% CO2 as a growing gas).23 Under these conditions there is also an increase of the MCRred2 form. In vitro experiments, the MCRred2 form can be induced by incubating the MCRred1 form with HS-CoM and HS-CoB (Figure 4).24 MCRred2 Ni(I) can be converted into MCRox1 Ni(III) through an oxidation step using polysulfide.25 It is not clear how this is done inside the cell, but when the gas phase is changed to 80% N2/20% CO2 to represent more oxidative condition, the MCRox1 form can be detected.23 The MCRox1 form is relatively stable in the presence of molecular oxygen. The red forms are highly sensitive to oxygen. The MCRred1 can be formed from MCRox1 by using strong reducing conditions.26 All of these MCR forms can be converted into a form that has the nickel in the 2+ oxidation state and are

10.1021/jp709860c CCC: $40.75 © 2008 American Chemical Society Published on Web 02/02/2008

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J. Phys. Chem. B, Vol. 112, No. 8, 2008 2467

Figure 1. Net reaction of MCR. The model system considered HSCH3 rather than HSCoB. The reaction is observed to be spontaneous by -7.2 kcal/mol. The present calculation give -3.2 kcal/mol for the model including HSCoB and -4.1 kcal/mol for the model including HSCH3.

therefore not detectable in electron paramagnetic resonance (EPR) spectroscopy. These EPR-silent forms are designated as for example “MCRox1-silent”. The form designated MCRsilent is a form that has been purified directly from cells. Three general mechanisms have been presented for the catalytic production of methane by MCR: (1) the Ni-Me/ Ragsdale pathway;21 (2) the Ni-Me/Thauer pathway;27 and (3) the methyl radical pathway.28,29 In the first two mechanisms, the methyl group from the CH3SCoM- cofactor oxidatively adds to the nickel center to form a Ni(III)-Me intermediate. The Ni-Me/Ragsdale pathway postulates the formation of a heterodisulfide radical in the initial phase, while the Ni-Me/Thauer pathway involves the transformation of CH3SCoM- into HSCoM-. Both mechanisms have been criticized because the forming Ni-Me bond is much weaker than the breaking S-Me bond (in CH3SCoM-),28 which would make that step in the cycle unrealistic. In the third mechanism, the methyl radical pathway, the S-Me bond cleaves into two radicals where methane is formed when the methyl radical abstracts a hydrogen atom from HSCoB. A recent discovery that bears on the mechanism is that bacteria that metabolize methane contain an enzyme that is very similar to MCR, which suggests that the mechanism which produces methane may be reversible and metabolize methane as well.30,31 Density functional calculations can now not only confirm proposed mechanisms but also be used to propose new ones.32,33 In this paper, we use DFT calculations to suggest a new mechanism for methane production from MCR.

Computational Methods All electronic-structure calculations were performed with Gaussian03.34 The structures were optimized using a hybrid DFT functional (B3LYP) with a mixed basis set consisting of 6-31G(d) for Ni/S and 3-21G for other atoms (BAS1). Single-point calculations were made with a larger mixed basis set, 6-311G(d) for Ni/S and 6-31G(d) for other atoms (BAS2). Solvation effects were included on geometries obtained at the B3LYP/ BAS1 level with CPCM and tesserae set to 0.10 Å2 (Table 1). In the conductor-like polarizable continuum model (CPCM),35 the solute molecule is placed into a cavity surrounded by the solvent considered as a continuum medium with a dielectric constant of 78.39 (water). The charge distribution of the solute polarizes the dielectric continuum, which creates an electrostatic field that in turn polarizes the solute. In specifying the molecular cavity, the united force field was used (i.e., RADII)UFF). The choice of UFF radii, which includes individual spheres for hydrogen atoms, was required when a hydrogen atom is being transferred (C-H-N, S-H-N, N-H-N). The experimental free energy of solvation was used for H3O+ (-110.2 kcal/mol).36 The computational model for MCR used the full F430 geometry with a tetrapyrrole (corphin) ring and all substituents. The models studied have charges which varied from +2 to -1. The HSCoM- cofactor (HSCH2CH2SO3-) and CH3SCoM(CH3SCH2CH2SO3-) were unprotonated while the five carboxy groups (two acetic acid groups and three propionic acid groups) on the corphin ring were all neutral (i.e., RC(O)OH). All of the groups had the correct stereochemistry on the F430 ring. In vivo, the F430 carboxy groups would interact with the amino acid

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TABLE 1: NPA Charges on Ni, Spin-Squared Values, Spin Densities on Ni, Total Energies (hartrees), and Solvation Free Energies (kcal/mol) for Species Related to Methane Production from MCR C/Ma CH4 H2O H3O+ HSCH3 CH3SHSCoMCH3SCoMSCoM2CH3SSCoMHSCoB2CoMSSCoB3MCRsilent MCRox1-silent Ni(II)F430-s Ni(II)F430-t Ni(III)F430-d Ni(III)F430-q MCRared1(Ni(I)F430) MCRbred1 MCRbsred1 MCRqred1 complex1a complex1b complex1c complex1d complex1d-TS F430zw F430zw-TS complex2 complex2-TS complex3 complex4a complex4b complex5 complex5-TSa complex5-TSb complex3-TSa complex6(MCRox1) complex3-TSb complex7 complex8 complex8-TS complex9 CH3 CH2CH2CH2SO3SCH2CH2SO3MCRBrMe MCRBPS

01 01 11 01 -1 1 -1 1 -1 1 -2 1 -1 1 -2 1 -3 1 -1 3 -1 3 11 13 22 24 02 02 02 04 12 12 12 12 12 02 02 02 02 02 -1 2 -1 2 02 02 02 02 02 02 02 -1 2 -1 2 -1 2 02 -1 2 -1 2 12 02

NPA(Ni)b

1.52 1.41 1.20 1.47 1.49 1.48 1.02 1.01 1.39 1.44 0.99 1.33 1.25 0.99 1.03 0.97 1.29 1.00 1.02 1.21 0.94 0.94 1.31 1.35 1.20 1.37 1.44 1.34 1.42 1.28 1.06 1.00

1.42 1.43

〈S2〉c

spin(Ni)d

2.00 2.01

1.71 1.62

2.00 1.65 3.77 0.91 0.89 1.57 3.77 0.82 0.79 1.22 0.81 0.86 0.83 1.39 0.78 0.77 0.81 0.76 0.75 1.70 1.70 1.26 1.41 1.24 0.84 0.84 1.33 0.84 0.88 0.75 0.75 0.75 0.84 0.99

1.71 1.66 1.73 1.11 1.09 1.58 1.73 1.03 0.97 1.38 1.00 1.08 1.02 1.46 0.98 0.95 1.04 0.02 0.01 1.56 1.53 1.29 1.49 1.40 1.11 1.07 1.43 1.07 1.11

1.07 1.25

B3LYP/BAS1e

B3LYP/BAS2 f,g//B3LYP/BAS1e

CPCM/BAS1g,h

-40.30160 -75.97396 -76.28196 -438.47798 -437.88949 -1099.61476 -1138.71537 -1098.92607 -1536.91064 -1746.69519 -2845.05061 -5982.98784 -5982.94645 -4883.71189 -4883.73861 -4883.42186 -4883.42057 -4883.86655 -4883.89798 -4883.86313 -4883.84623 -4884.26750 -4884.27654 -4884.27377 -4884.27398 -4884.25983 -4883.89180 -4883.83898 -6023.12143 -6023.09207 -6023.10166 -6461.04608 -6461.06507 -4924.12180 -4924.11111 -4924.05525 -6023.04271 -5982.84310 -6023.08709 -6023.13950 -5983.53057 -5983.50745 -5983.49057 -39.62224 -739.85992 -1098.98444 -4923.39251 -5623.73158

-40.51766 -76.40569 -76.68370 -438.72410 -438.15007 -1101.33839 -1140.65124 -1101.34203 -1538.87320 -1752.26315 -2852.33801 -6003.29312 -6003.27530 -4902.33491 -4902.35773 -4901.99963 -4902.02061 -4902.50879 -4902.51624 -4902.49218 -4902.46776 -4902.89807 -4902.90205 -4902.88344 -4902.88499 -4902.87004 -4902.49066 -4902.44088 -6043.63670 -6043.60065 -6043.60962 -6481.81869 -6481.83716 -4942.93135 -4942.92300 -4942.87003 -6043.54350 -6003.15572 -6043.57981 -6043.66563 -6003.85952 -6003.81261 -6003.82597 -39.83752 -741.76424 -1100.70439 -4942.22290 -5644.22880

-0.21 -7.14 (-2.84)i -83.95 (-110.2)j -3.68 -78.13 -64.43 -66.12 -60.81 -63.99 -176.85 -282.80 -110.12 -111.53 -79.85 -78.20 -154.25 -160.89 -53.83 -50.53 -58.71 -56.97 -80.81 -78.75 -78.41 -76.67 -74.25 -47.39 -52.36 -71.49 -70.75 -73.10 -109.66 -98.16 -49.67 -49.51 -49.43 -69.92 -77.05 -69.12 -76.09 -104.52 -96.46 -106.70 -0.60 -66.02 -62.32 -77.88 -78.52

a Charge and multiplicity. b Natural population charge (NPA) charge on nickel at the B3LYP/BAS2 level. Reed, A. E.; Curtiss, I. A.; Weinhold, F. Chem. ReV. 1988, 88, 899. c Spin-squared 〈S2〉 value at the B3LYP/BAS2 level. d Unpaired spin density on Ni at the B3LYP/BAS2 level. e BAS1 ) 6-31G(d) basis for Ni and S; 3-21G for all other atoms. f BAS2 ) 6-311G(d) for Ni and S; 6-31G(d) for all other atoms. g The keyword SCF)TIGHT was used for all single-point energy calculations. h For CPCM calculations, the solvent was water ( ) 78.39) and the keywords RADII)UFF and TSARE)0.1 were used and (occasionally) OFAC ) 0.8 and RMIN ) 0.5 were required. The values reported in this column do not include a correction factor of 1.9 kcal/mol that was added to account for the change in state from 1 atm at 273 K to 1 M at 298 K. i A factor of 2.4 kcal/mol was added to account for the change of state from 1 M (solution) to 55.5 M (liquid water). j Experimental value. Reference 36.

residues of MCR. However, in the present model, surrounding residues are absent. As noted below, one structure (MCRbrad1) was characterized by internal hydrogen bonding of the carboxy groups. The interaction was stabilizing in the gas phase, but much less so when the effects of solvation were included. The protein environment has a variable dielectric depending on the number of nearby polar groups with estimates ranging from ) 2 to 10.37 We chose to include a fraction of calculated aqueous solvation. Test calculations suggested taking 2/3 of the aqueous solvation would approximate a dielectric of about 4. However, when we compared the experimental cyclic voltamogram of NiF430, we used the full aqueous solvation energies. Our estimates of relative energies combine the electronic energies differences at the B3LYP/BAS2//B3LYP/BAS1 level

with 2/3 of solvation calculated at the CPCM/B3LYP/BAS1 level (eq 1).

∆G(aq) ≈ ∆E(gas)/BAS2 + (2/3)∆G(solvation)/BAS1 (1) We understand that combining electronic energies and aqueous free energies is not strictly correct, but we feel that the comparison provides an estimate of relative stabilities of different structures within the protein environment. Thus, we will not distinguish between enthalpies and free energies when discussing relative energies.38 In the figures that follow, only a portion of the F430 model will be shown to highlight the geometric changes under discussion. To aid in visualization, the orientation of the F430 cofactor will be maintained (i.e., the

Methane Production


TABLE 2: Comparison Distances, Spin Densities, and NPA Charges of MCRsilent, MCRox1-silent, MCRared1, MCRbsred1, complex1b, and complex1aa MCRsilent NiNA-ring NiNB-ring NiNC-ring NiND-ring NiH NC-ringH Ni-O

2.065 2.066 2.120 2.020

Ni NA-ring NB-ring NC-ring ND-ring HNi/HNC-ring

1.71 (1.52) 0.06 (-0.56) 0.05 (-0.64) 0.04 (-0.69) 0.06 (-0.60)

MCRox1-silent 2.118 2.045 2.101 2.055

1.62 (1.41) 0.05 (-0.53) 0.05 (-0.64) 0.05 (-0.67) 0.04 (-0.62)

MCRared1

MCRbsred1

Distance (Å) 2.245 1.908 2.019 1.963

2.231 1.981 2.067 2.037

4.230

2.088

Spin (NPA charge) 1.11 (1.02) 0.03 (-0.56) 0.05 (-0.68) 0.08 (-0.63) 0.04 (-0.67)

1.09 (1.01) 0.03 (-0.57) 0.04 (-0.66) 0.08 (-0.63) 0.03 (-0.67)

complex1b 2.210 1.981 2.103 1.436

0.97 (1.33) 0.05 (-0.55) 0.02 (-0.60) 0.07 (-0.65) 0.02 (-0.59) -0.14 (-0.09)

complex1a 2.025 1.899 2.505 1.955 2.095 1.062

1.03 (0.99) 0.05 (-0.60) 0.04 (-0.67) 0.02 (-0.62) 0.02 (-0.62) 0.00 (0.45)

a The notations N A-ring, NB-ring, NC-ring, and ND-ring indicate the ring nitrogen in the corphin ring. See Figure 2 for numbering. The B3LYP/BAS2 method was used to compute spin densities and NPA charges. A negative spin signifies excess β spin electrons.

Figure 3. Molecular plot F430 in the MCRox1-silent form (pdb 1HBN) with the SCoM2- ligand. The HSCoB cofactor is also shown above.

involved in the catalysis. We start by providing a molecular description for several of these species. Figure 2. The numbering system for cofactor F430. The ligand OdCH2CH2C(H)NH2C(H)O (surrogate for glutamine, Gln147) is shown coordinated to the Ni center. The molecular plot of the model system is show below in the same orientation.

C-ring will always be to the right). Full Cartesian coordinates of all complexes listed in Table 1 are given in Supporting Information. The primary motivation of this work is to provide a mechanism for the production of methane by MCR. This enzyme has received extensive study, and a number of intermediates have been characterized, some of which are not

Results and Discussion Molecular Description of Known MCR Forms. MCRsilent. An X-ray structure has been determined where the Ni(II) center is coordinated through an oxygen of the sulfate group to CoMSSCoB.22 A model of MCRsilent was constructed with the SCoM2ligand coordinated to the Ni(II) center, which is an isomer of MCRox1-silent where the SCoM2- ligand is coordinated to the Ni(II) through the thiol sulfur. Including 2/3 solvation, the MCRsilent form is 10.2 kcal/mol more stable than MCRox1-silent. In both forms, the Ni center is 6-coordinate (Figure 5). In


Duin and McKee

Figure 4. The experimental relationship between MCRred1, MCRred2, MCRox1, and MCRox1-silent is shown above. The calculations were performed on the species shown.

MCRsilent, one of the oxygens of the sulfonate group is hydrogen bonded to the lactam ring of F430 with a short OsHN distance of 1.744 Å. MCRox1-silent. The MCRox1-silent form contains a bound SCoM2- with a nearby HSCoB cofactor.22 MCD evidence shows that the structure has a high-spin (triplet) Ni(II) center.39 The calculated Ni-N distances are very similar to the X-ray distances (Figure 5). The calculated Ni-S distance of the SCoM2- ligand and the Ni-O distance of the glutamate are about 0.1 A longer than distances obtained by experiment (2.482 vs 2.396 Å, Ni-S; 2.487 vs 2.350 Å, Ni-O). The geometry of MCRox1-silent with the SCoM2- ligand removed was optimized as a singlet and triplet (Figure 6, Ni(II)F430-s and Ni(II)F430-t). The singlet spin state contains two electrons in the dz2 orbital which prevents the close approach of the Gln147 ligand (Ni-O, 2.458 Å) which is still held by the F430 cofactor through hydrogen bonding. On the other hand, the triplet spin state has only one electron in the dz2 orbital and the oxygen of the glutamine approaches the Ni(II) center to 2.020 Å. Another geometric different between the singlet and triplet states of MCRox1-silent is the Ni-N distance to the nitrogen atoms in the corphin ring. The “extra” electron in the dx2-y2 orbital increases all of the Ni-N distance for the triplet. The calculated triplet R spin density on Ni is 1.71 and the NPA

charge is 1.47, both consistent with Ni(II). The triplet is 13.2 kcal/mol more stable than the singlet. MCRox1. The MCRox1, MCRred1, MCRred2 species are EPRactive forms, thought to involve Ni(I) and/or Ni(III) oxidation states.40-43 The three forms can interconvert as shown in Figure 4, where the MCRred1 form is thought to be the active catalytic species.24,25 The MCRox1 form (complex6) is known from EXAFS data to be a Ni(III) 6-coordinate complex with distances to the axial SCoM2- ligand of 2.40 Å (Ni-S) and to the axial glutamine ligand of 2.08 Å (Ni-O).44,45 Our values of 2.352 and 2.066 Å, respectively, are in good agreement. A comparison of computed spin densities and NPA charges for MCRsilent and MCRox1-silent (both Ni(II) species and MCRox1 (a Ni(III) species) species reveals (Table 2) that the charge on the Ni center changes very little. For example, the charge on Ni increases only 0.03e- from MCRox1-silent to MCRox1 (Ni(II) f Ni(III)). The atom undergoing the largest change in charge is the thiol sulfur of the SCoM2- ligand (+0.36e-). There is significant spin polarization in the B3LYP calculation of MCRox1 as shown by the large deviation of the 〈S2〉 value (1.24 found, 0.75 expected). The computed spin densities are in line with XAS measurements that show almost identical K-edge positions for the MCRox1, MCRox1-silent, and MCRsilent forms.44,45

Methane Production


Figure 5. A comparison between the X-ray structures of the active site of MCRsilent (pdb 1MRO) and MCRox1-silent (pdb 1HBN) on the left-hand side and the calculated structures on the right-hand side. Hydrogens are omitted for clarity. The coordination environment around Ni is also compared.

Figure 6. The optimized structures of the singlet and triplet spin states of F430 without the SCoM2- ligand are shown above. The d-orbital orbital occupation for the singlet has the dz2 doubly occupied, while the triplet has one electron in dx2-y2 and dz2.

MCRred1. The active form of MCR is the MCRred1 form. It can be made to accumulate in the cells by using a more reducing

environment (100% H2).23 The oxidation state of nickel is Ni(I) with unpaired spin density in dx2-y2. The Ni center has


Duin and McKee

Figure 7. The glutamine-surrogate in the “a” form of MCRred1 (MCRared1) is held through hydrogen bonding with carboxy groups linked to the C8 and C13 positions. In the “b” form (MCRbred1), the carboxy group linked to C13 forms strong hydrogen bonding with the carboxy group linked to the C8 position rather than to the glutamine residue. In the Ni-protonated form of MCRred1, the glutamine-surrogate is held through the hydrogenbonding pattern as in MCRared1.

been shown to be 5-coordinate.44 (Ragsdale and co-workers proposed that there is a very weak, easily exchangeable, sixth ligand present.45) In the presence of HSCoM- and HSCoB, MCRred1 can interconvert with another reduced form called MCRred2.24 Recently it was shown that there are two forms present in this state, MCRred2a (axial) and MCRred2r (rhombic), that are in thermal equilibrium. At low temperature (0-4 °C), MCRred2a is preferred and at high-temperature (>25 °C) a mixture of both forms can be detected.46 We calculated two conformations of MCRred1 (MCRared1 and MCRbred1). In form “b”, there are strong internal hydrogen bonds involving two carboxyl groups linked to the F430 cofactor at the C8 and C13 positions (see Figure 7). Form “a” does not have the internal hydrogen bonds and is less stable than “b” by 4.7 kcal/mol in the gas phase, 2.5 kcal/mol with 2/3 solvation, and only 1.0 kcal/mol with full solvation. Since the carboxy groups of the actual F430 cofactor are much more likely to interact with the surrounding residues, we chose form “a” as our standard reference. We did not seek other minima with

internal hydrogen bonds involving carboxyl groups linked to the C8 and C13 positions. In addition to MCRared1, another form (“broken spinsymmetry”, MCRbsred1), with a much larger 〈S2〉 value (MCRared1/MCRbsred1, 0.91 and 1.57, respectively) and only 7.1 kcal/mol less stable, was located. The MCRbsred1 “broken spin-symmetry” form can be best described at a triplet Ni(II) center low-spin coupled with a radical anion corphin ring (Ni(II)8(π*)1).47 In Figure 8 and Table 2, a comparison of the geometry and R/β spin densities is made for MCRared1, MCRbsred1, and MCRqred1. The MCRared1 structure is 4-coordinate due to a (dz2)2 Ni occupation which is repulsive to ligands in the axial position. On the other hand, the MCRbsred1 form has a (dz2)1 Ni occupation which allows the glutamine to coordinate (Ni-O distance is 2.088 Å) and the Ni center to become 5-coordinate. As already mentioned, EXAFS studies showed that MCRred1 is 5-coordinate.44 Thus, even though MCRbsred1 is calculated to be 7.1 kcal/mol less stable than MCRared1, the true MCRred1 structure may be a hybrid of

Methane Production


Figure 8. Three forms of MCRred1: form “a” d9-doublet, form “bs” broken spin-symmetry d8-doublet, and form “q” d8-quartet state. The experimental form of MCRred1 is known from EXAFS to be 5-coordinate around the Ni center.

Figure 9. Structures of the doublet and quartet spin state of Ni(III)F430 (right) and comparison with the Ni(I)F430 (left). The lowest-energy species (quartet state) is expected from the one-electron oxidation of Ni(II)F430.

the two structures such that the dz2 orbital occupation is sufficiently decreased from two to allow the glutamine ligand to coordinate. The quartet spin state, which corresponds

to a high-spin coupling of the Ni(II) center to the corphin radical anion (Figure 8), was 8.6 kcal/mol less stable than MCRared1.


Duin and McKee

Figure 10. Catalytic cycle of F430 showing the production of methane from CH3SCoM- and HSCoB. On the bottom are the relative energies in kcal/mol at the B3LYP/BAS2//B3LYP/BAS1 + 2/3‚CPCM/B3LYP/BAS1 level. The formal oxidation state of Ni is indicated for the various intermediates inside the catalytic circle. The physical oxidation states of these intermediates are close to Ni(II). The overall reaction difference is -6.0 kcal/mol. This value is different from the -4.1 kcal/mol value reported in Figure 1 because the former used full solvation and the latter used 2/3 solvation (see text). The dotted line indicates the regeneration of the active catalyst complex1a via protonation of Ni(I)F430 which is modeled by the reaction H2O + HSCH3 f H3O+ + SCH3-. In the present mechanism of MCR, the HSCoB cofactor is deprotonated, but the protonation of Ni(I)F430 takes place by protonation of the amide side group of Gln147 and subsequent proton transfer to the C-ring nitrogen of Ni(I)F430. Thus, the unfavorable energy change required to complete the catalytic cycle (50.8 kcal/mol) may occur via many steps rather than a single step which would be unrealistic.

MCRred2. Recent work by Kern et al.48 showed that what we used to call the MCRred2 form actually consists of two species with different EPR signatures: axial (MCRred2a) and rhombic (MCRred2r). We have computed several structures which may correspond to these forms. These results will be communicated in a later publication together with the full spectroscopic characterization of these forms by the group of Dr. Jeffrey Harmer using ENDOR spectroscopy. Computed Redox of F430 (Ni(I)/Ni(II)/Ni(III)). Jaun and coworkers50,51 measured the cyclic voltammogram of Ni(II)F430M (M indicates the five carboxy groups of F430 are replaced with methyl ester groups). Relative to the standard hydrogen electrode (SHE), the Ni(I)/Ni(II) couple is -0.65 V and the Ni(II)/Ni(III) couple is 1.49 V. Within the protein, an irreversible conversion of MCRred1 into MCRsilent was observed with an Em of -0.44 V (pH 7.2, vs SHE).25

The calculations used the Ni(I)F430 model with a glutaminesurrogate in the axial position with full aqueous solvation, while the experimental studies would included a solvent molecule (propionitrile) in that position. The Ni(I)F430 form is identical to our MCRared1 where the electronic description is (dx2-y2)1(dz2)2 with a contribution from a doublet-coupled (dx2-y2)1(dz2)1(π*)1 configuration. Electron loss from Ni(I)F430 produces a (dx2-y2)1(dz2)1 triplet. The next electron is removed from a nitrogen lone pair of the glutamine ligand, where the quartet spin state is 17.6 kcal/mol lower than the broken spin-symmetry doublet state. The energy difference for Ni(I) f Ni(II) + e- (3.05 eV) is referenced with the calculated free energy change (-4.34 eV) for the standard SHE (1/2H+ + e- f 1/2H2(g)),52 to give an oxidation potential of -1.29 V, which can be compared with an experimental value of -0.65 V. The computed energy different for Ni(II) f Ni(III) + e- is 5.59 eV which yields an

Methane Production


Figure 11. Three structures obtained from the protonation of MCRared1. Complex1a is protonated on the C-ring nitrogen and is characterized by a normal doublet spin-squared value of 0.82. Complex1b is protonated on the Ni center. Complex1c is protonated on the C-ring nitrogen and is characterized by a large deviation from the expected doublet spin-squared value (1.22). The three forms are protonated spontaneously (relative to MCRared1 plus H3O+) by -16.2, -17.4, and -5.5 kcal/mol, respectively.

oxidation potential of 1.25 V, which is close to the experimental value of 1.49 V. The calculations indicate the stability of the Ni(II) oxidation state. In fact, when Ni(II)F430 is oxidized, the electron largely comes from the more distant nitrogen lone pair of the glutamine ligand. That particular nitrogen shows an unpaired spin density of 0.34 R electrons. In turn, the electron added to Ni(II)F430 partially goes into the π* orbital of the corphin ring as shown by significant unpaired R and β spin density on the ring (Figure 9). While the formal oxidation state of nickel changes by one unit in each oxidation step, Ni(I) f Ni(II) f Ni(III), the calculated NPA charges on Ni change much less (NPA charge, Ni(I) ) 1.02 f Ni(II) ) 1.47 f Ni(III) ) 1.48). Thus, while the formal oxidation state changes by 1 unit, the physical oxidation state changes much less.53 If fact, the “Ni(II) f Ni(I)” reduction can be viewed as adding one electron to the corphin π system, and the “Ni(II) f Ni(III)” oxidation as removing one electron from a ligand lone pair. Catalytic Cycle. The catalytic cycle for production of methane is shown in Figure 10. A key discovery in the present work is the facile protonation of Ni(I)F430. Two protonation sites

were almost equally favored (Figure 11). Protonation (addition of H3O+ and elimination of H2O) on nickel produced a Ni(III) center. The doublet (complex1b) contains one electron in a dx2-y2 orbital and an empty dz2 orbital (Figure 11). Coordination of the glutamine-surrogate to the d7 system is favorable as shown by the calculated Ni-O distance of 2.033 Å. On the other hand, protonation can occur on the C-ring nitrogen of corphin. The resulting nickel is Ni(I), a d9 system with one electron in a dx2-y2 orbital. In this case, coordination of the glutamine-surrogate is unfavorable, but it remains in the vicinity through hydrogen bonding to the carboxy groups linked to the C3 and C8 positions of the corphin ring. It has to be noted that in the enzyme-bound form the two oxygens of the C3 carboxy group (COO- form) can H-bond to the nitrogen of the backbone amide of Val145, Val146, and/or Gln147.27 The C8 carboxy group is too far away to H-bond to the Gln147. Although our model introduces this artificial H-bonding pattern, it is still a good mimic of the situation present in the active site where the side-chain carbonyl of Gln147 is kept in close range of the nickel of F430.27 The protonation at Ni is spontaneous by 17.4 kcal/mol while protonation of the corphin ring is spontaneous by 16.2 kcal/ mol. In the ring-protonated form, the Ni-H hydrogen distance


Duin and McKee

Figure 12. The “d” form of protonated MCRared1 (complex1d) is protonated on the amino side-chain of the glutamine residue (Gln147). The transition state (complex1d-TS) for transfer of the proton to the C-ring nitrogen to form complex1a has a barrier of 11.0 kcal/mol (22.0-11.0). The zwitter-ionic form of MCRared1 (F430-zw) is higher in energy than MCRared1 by 18.1 kcal/mol. Relative to F430-zw, the transition state (F430zwTS) for transferring a proton to the C-ring nitrogen is 28.0 kcal/mol higher (46.1-18.1).

to Ni is 2.095 Å, which indicates some agostic interaction (the corresponding Ni-N distance is 2.505 Å). Another protonated form (spontaneous by 5.4 kcal/mol) was found (complex1c) where the hydrogen (attached to the C-ring nitrogen) extends away from the glutamine coordination site. The significant deviation from the expected spin-squared value (1.22 calculated, 0.75 expected) indicates the complex can be viewed as a Ni(II) center coupled with an unpaired electron on the ligand. The mechanism starts with the protonation of Ni(I)F430 to form complex1a where the pKa is estimated to be between 4 and 12.54 Access of bulk solvent to the Ni(I)F430 active site is known to be blocked by the HSCoB cofactor. However, there are two water channels present in MCR that connect the active site to the bulk of the water on the outside.22,27,55 One channel ends up in the active site channel at the site in the crystal where the coenzyme M sulfonate group is bound. The second channel ends at the amido group of the Gln147 residue on the distal side of F430. We believe that the side-chain amido group of Gln147 may provide a proton relay to the C-ring nitrogen. In the X-ray structure of MCRox1-silent, the amino side group of Gln147 is

in close proximity to the C-ring N (H2N-Nc, 3.39 Å). If the amino side group is protonated, that proton might be transferred to the C-ring nitrogen. We computed the energy of our model protonated on Gln147. The energy of complex1d is 11.0 kcal/ mol higher than our C-ring N-protonated model. Two of the hydrogens on the NH3 group are strongly H-bonded to the carboxy groups linked to the C8 and C13 positions while the third hydrogen is 2.300 Å from the C-ring nitrogen. A transition state (complex1d-TS) for proton transfer was located 22.0 kcal/ mol above complex1a. Thus, if the amido group of the Gln147 side chain is protonated, that proton can easily be transferred to the C-ring nitrogen. Again it has to be noted that the situation in the enzyme environment is different. In the crystal structures a water molecule is present (pdb 1MRO; HOH7791) that is H-bonded to both the C13 carboxy group (COO- form) and the side-chain amido group of Gln147. This is the water molecule that forms the end of the second water channel on the distal side of F430. An alternative mechanism was considered where an internal shift of a proton occurs from the carboxy group (linked to the C8 position) to Gln147. This zwitterionic species F430zw is 18.1

Methane Production

Figure 13. The complex between SCoM2- and MCRared1 (complex2) is held through hydrogen bonding. In the transition state for oxidative addition (complex2-TS), the breaking S-C bond and making Ni-C/ Ni-S bonds are 2.182, 2.204, and 2.212 Å, respectively. The Ni center in the product complex (complex3) is out of the plane formed by the four corphin nitrogens,

kcal/mol less stable than Ni(I)F430 (also known as MCRared1). An estimate of the transition state (F430zw-TS) was made by fixing the two N-H distances to 1.30 Å and optimizing the remaining geometric parameters. The resulting structure was 28.0 kcal/mol (46.1-18.1) higher than the zwitterionic species F430zw. In our mechanism, we favor the initial protonation of the Gln147 group followed by the transfer of that proton to the C-ring nitrogen of the Ni(I)F430 cofactor to form complex1a (Figure 12). The protonated Ni(I)F430 cofactor (complex1a) can complex CH3SCoM- to form complex2. We predict the association energy to be -5.8 kcal/mol with a Ni-S distance of 3.082 Å. Two oxygens of the SO3 group form hydrogen bonds with CH groups of F430 (see Figure 13, 1.818 and 1.854 Å). In the oxidative addition step (complex2 f complex3), the S-Me distance becomes 2.182 Å and the Ni-S distance becomes 2.212 Å. The activation barrier is 23.1 kcal/mol (17.3 + 5.8) with respect to complex2. In the product complex (complex3), the Ni has moved a considerable distance (1.5 Å) out of the approximate plane formed by the four nitrogen atoms of the corphin ring where the tetrapyrrole ring itself is not distorted


Figure 14. The CH3S- group is fixed 7.0 Å from the four nitrogens of corphin (complex4a). If the CH3S- group is allowed to optimize, complex4b is obtained, which is 3.9 kcal/mol more stable. The S∴S interactions in complex4a and complex4b are 2c-3e interactions.

to any degree. The Ni atom is now coordinated by Me, SCoM2-, and two of the four nitrogen atoms in the corphin ring (Figure 13). Complex3 is 10.1 kcal/mol less stable than complex1a + CH3SCoM-. In the next step, a CH3S- group was allowed to optimize while fixing the four distances to the nitrogen atoms of the corphin ring to 7.0 Å (Figure 14). The CH3S- represents a deprotonated HSCoB (i.e., SCoB-) and the fixed 7.0 Å distances mimic the fact that the HSCoB group is constrained in how close it can approach the Ni(I)F430 cofactor. The constrained system is 10.6 kcal/mol more stable than separated species. It is interesting to point out that two-center three-electron (2c-3e) bonding is very common between two sulfur centers when charge and spin delocalization are possible.56 This is the case in the constrained complex4a where unpaired spin density on the sulfur centers is significant (Figure 14). The unpaired spin density on Ni is only 0.02 (Table 1) which indicates that the nickel is low-spin Ni(II) with the unpaired spin density located almost entirely over the sulfur centers (total spin density over two sulfurs ) 0.95 R). It is possible that the strength of 2c-3e bonding is overestimated in this model because density functional theory is known to overestimate spin/charge delocalization.57 Thus, the S∴S distance is very long (3.720 Å), yet the binding of SCH3- to complex3 is 10.6 kcal/mol. The Ni-S distance in the complex has increased only 0.04 Å (complex3


Figure 15. Complex5 contains a coordinated methyl group with a very accessible transition state (complex5-TSa) to abstract a proton from the C-ring nitrogen to form methane. The transition state for elimination of methane with inversion of stereochemistry (complex5TSb) is 33.2 kcal/mol higher than than for complex5-TSa.

f complex4, 2.170 f 2.211 Å) while the Ni-CH3 distance has decreased 0.05 Å (1.963 f 1.914 Å). If the constraints on the four S-N distances are removed and the complex is allowed to fully optimize, the complex is stabilized an additional 3.9 kcal/mol and the S∴S distance decreases to 2.957 Å (Figure 14, complex4b). This aspect may explain one of the enigmas of MCR catalysis. If the methylene chain linker in HSCoB is decreased by one CH2 group, MCR activity is reduced and if the linker is increased by one, MCR activity is also reduced.58 The HSCoB linker may be optimized to interact with the SCoM2- ligand but not allow the HSCoB to form a strong Ni-S interaction. It is also worth noting that the out-of-plane distortion of Ni from the approximate plane of the four nitrogen atoms of corphin allows the sulfur of the SCoM2- ligand to extend much closer to the HSCoB cofactor than would otherwise be possible. By binding CH3S-, the Ni in complex3 is reduced from Ni(III) to Ni(I) (via a Ni(II) center on complex4a) where the CH3S- anion effectively transfers two electrons to the Ni center. The CH3SSCoM- radical anion ligand in the constrained complex (complex4a) is bound by 7.5 kcal/mol with respect to complex5, where Ni is now 4-coordinate. The loss of the CH3SSCoM- ligand is compensated by the recoordination of

Duin and McKee

Figure 16. Starting from complex3, an alternative pathway to form MCRox1 (complex6) is possible through transition state complex3-TSa.

the D-ring nitrogen to the Ni center (Figure 15). The out-ofplane distortion of the Ni is much less, and the hydrogen atom bonded to the C-ring nitrogen is 2.007 Å from Ni. From complex5, a very low energy pathway exists for loss of CH4. The Ni-HN(C-ring) distance decreases from 2.007 to 1.783 Å and the C-ring nitrogen approaches to 2.428 Å in the transition state (complex5-TSa). In addition, the Ni-C distance increases from 1.989 to 2.146 Å and the breaking H-N and making C-H distances are 1.169 and 1.638 Å (Figure 15). The reaction forms Ni(I)F430 in a step that is spontaneous by 63.8 kcal/mol (Figure 10). The elimination of methane via complex5-TSa occurs with retention of stereochemistry. However, inversion of stereochemistry was observed when the EtSCoM- cofactor was used.57 For that reason, we located a second transition state complex5TSb where methane was eliminated with inversion of stereochemistry at carbon. The Ni-C bond is 0.183 Å longer than that in complex5-TSa and the CH3 moiety is nearly planar. While complex5-TSb appears to have more room for the migrating group and might better accommodate an ethyl group than complex5-TSa, the additional 33.2 kcal/mol (45.6 - 12.4) of required activation would make competition unlikely. Thus, the proposed mechanism is in line with the available spectroscopic and kinetic data, but no inversion is predicted about the carbon center. While studies with the labeled substrate analogue ethyl-coenzyme M (EtSCoM-) indicated that the catalytic

Methane Production


Figure 17. Starting from complex3, an alternative pathway to form complex6 is possible through transition state complex3-TSb.

reaction proceeds with inversion of stereochemistry on carbon,59 the observed activity with this substrate is 1% of the activity observed with the natural substrates. With such a big difference it is possible that the mechanism followed for the reaction of EtSCoM- is different from the one followed for CH3SCoM-. There is evidence that the MCR protein can operate in both directions, i.e., methane can be formed in some organisms and methane can be consumed in other organisms. The large increase in stability upon CH4 formation presents a problem for the reversibility of the reaction. We propose (without computational evidence) that loss of CH4 is simultaneous with the re-formation of protonated Ni(I)F430. In that case the reaction complex5 + H3O+ f complex1a + CH4 + H2O is spontaneous by 13.0 kcal/mol. Our mechanism is completed by protonation of Ni(I)F430 by H3O+ which, in turn, is coupled with the deprotonation of CH3SH (surrogate for HSCoB). The question of how HSCoB is deprotonated and how Ni(I)F430 is reprotonated in the protein environment is a more complex question that is not addressed in this study. The energy for deprotonation of HSCoB in the enzyme environment will definitely be smaller since a mainchain nitrogen from Val482 and a side-chain nitrogen from Asp481 are both donating hydrogen bonds onto the coenzyme

SCoB- sulfur which would stabilize the negative charge. Future calculations should include these important hydrogen bonds. The proton for protonation of F430 in the enzyme should come from the water molecule that is present in the water channel present at the distal site of the ring. Alternative Reactions. We considered two alternative reactions starting from complex3. Rather than add CH3S-, complex3 can lose methane (Figure 16). The transition state (complex3-TSa) for this step was estimated by fixing the C-H and N-H distances to 1.50 and 1.25 Å, respectively, and optimizing the remaining parameters. The high activation barrier for this step (53.7 kcal/mol) strongly suggests that the addition of SCH3- (followed by loss of CH3SSCoB-) precedes methane formation. Another possible reaction that complex3 can undergo is the elimination of HSCoM- where the SCoM2- ligand abstracts the proton attached to the C-ring nitrogen (Figure 17). The transition state (complex3-TSb) was estimated by fixing the S-H and N-H distances to 1.60 and 1.30 Å, respectively, and optimizing the remaining parameters. The activation barrier for this process is 21.4 kcal/mol, and the product of the reaction (complex7) is more stable by 37.1 kcal/mol. The reaction complex3 f complex3-TSb f complex7 illustrates the


Duin and McKee

Figure 18. Oxidative addition of HSCoM- to MCRared1 takes place through transition state complex8-TS to form complex9. Values of species in the potential energy diagram are in kcal/mol.

changes in coordination environment experienced by the Ni center (Figure 17). The Ni center is 4-coordinate (distorted tetrahedral) in complex3, 5-coordinate in complex3-TSb (trigonal bipyramid), and 6-coordinate (octahedral) in complex7. Oxidative Addition of HSCoM. The MCRred2 species is formed by the addition of HSCoM- and HSCoB to MCRred1 (Figure 4). ENDOR studies using H33SCoM- showed that this compounds is bound directly to the nickel via the thiol sulfur.60 Here we postulate the oxidative addition of HSCoM- in the formation of the MCRred2r species. When the HSCoM-/ MCRared1 complex is optimized (complex8) and 2/3 aqueous solvation is included, the complex is 0.2 kcal/mol less stable than MCRared1 + HSCoM-. The transition state (complex8TS) is estimated by fixing the S-H and N-H distances to 1.60 and 1.30 Å, respectively, and optimizing the remaining parameters. The activation barrier is 34.8 kcal/mol, compared to 23.1 kcal/mol for the oxidative addition of CH3SCoM- (complex2 f complex3). The reaction product (complex9) is one of the candidate structures for MCRred2r. MCRBrMe and MCRBPS. As already mentioned, the formation of the Ni-Me species in the Ni-Me/Ragsdale pathway and the Ni-Me/Thauer pathway was not considered to be a realistic

step. This also put doubt about the formation of such a species per se. Recent work with the inhibitors (3-bromopropane)sulfonate (BPS) and bromomethane (BrMe) showed that a Ni-C bond is formed in the MCRBPS form and a Ni-CH3 species in the MCRBrMe form.46,61-63 The MCRBPS species formed is structurally similar to the MCRox1 form where the SCH2CH2SO32ligand is replaced by CH2CH2CH2SO32-. The addition of BrCH2CH2CH2SO3- and BrMe to MCRared1 produces the new species plus a bromide anion. The MCRBrMe and MCRBPS species have a Ni(III) center. The calculated homolytic bond energies are very similar for the three species (Figure 19). Siegbahn and co-workers28 computed a value of 18.0 kcal/mol for the methyl binding energy (eq 2) which is identical to our value of 18.0 kcal/mol.

CH3• + Ni(II)F430+ f CH3-Ni(III)F430+

(2)

Schofield and Halpern64 determined the experimental homolytic Ni-C bond dissociatiation energy of benzyl in a Ni complex in acetonitrile to be 19 kcal/mol, which is very similar to the values calculated for MCRBrMe (18.0 kcal/mol) and MCRBPS (24.5 kcal/mol).

Methane Production


Figure 19. Comparison of computed structures for MCRox1, MCRMe, and MCRBPS. The homolytic bond energies used eq 1.

Reversibility of Methane Formation. One advantage of the present mechanism for the reversible action of MCR is the fact there is a specific cleavage/formation of the C-H bond. As opposed to other mechanisms in which the CH3 radical abstracts a hydrogen atom or CH3 is protonated by solvent, in the present mechanism the formation/cleavage step of C-H should not be strongly affected by entropy. Conclusions The present study presents the most complete model of the F430 cofactor with a coordinated glutamine reported. Within this model, several structures of known MCR species have been presented. These include MCRsilent, MCRox1-silent, MCRox1, and MCRred1. In addition, two species have been included in this studywhicharerelevant.ThesearetheCH3- andCH2CH2CH2SO32ligands attached to the Ni(III)F430 cofactor (with a glutaminesurrogate in the distal position). They are the products from the reaction of BrMe and BrCH2CH2CH2SO3- with Ni(I)F430. When MCR is reduced to Ni(I), there is considerable unpaired spin density on the corphin ring. The charge and spin density on the Ni center only moderately change from that found in Ni(II) complexes. The production of methane from CH3SCoM- + HSCoB f CH4 + CH3CoSSCoB- in the MCR catalytic cycle starts with the protonation of MCR, either on the Ni center or on the C-ring nitrogen of corphin. The two forms are nearly equally stable. The next step is the oxidative addition of CH3SCoM-. The coordination around the center is substantially distorted, and the Ni adopts a position above the four nitrogen atoms of the corphin ring. A sulfur of the deprotonated HSCoB (SCoB-), which we model with a constrained CH3S- unit, is predicted to interact with the sulfur of the SCoM2- ligand to form a 2c-3e interaction. Elimination of CH3SSCoM-, leaves a CH3-substituted Ni with a hydrogen on the nitrogen of the corphin C-ring. A very low-energy transition state regenerates F430.

Acknowledgment. We thank Ruldolf Thauer, Bernhard Jaun, and Jeffrey Harmer for helpful discussions. This work was supported by the Petroleum Research Fund (E.C.D.) The Alabama Supercomputer Center is acknowledged for a generous grant of computer time. Supporting Information Available: Optimized Cartesian coordinates of all species in Table 1 and full citation for ref 34. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Conrad, R. Microbiol. ReV. 1996, 60, 609. (2) Thauer, R. K. Microbiol. 1998, 144, 2377. (3) Ellefson, W. L.; Wolfe, R. S. J. Biol. Chem. 1981, 256, 4259. (4) Ragsdale, S. W. Biochemistry of methyl-CoM reductase and coenzyme F430. In Porphyrin Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Elsevier Science: San Diego, 2003; pp 205-228. (5) Ermler, U. Dalton Trans. 2005, 3451. (6) Duin, E. C. Role of coenzyme F430 in methanogenesis. In Tetrapyrroles: their birth, life and death; Warren, M. J., Smith, A., Eds.; Landes Bioscience: Georgetown, 2008; in press. (7) Jaun, B.; Thauer, R. K. Methyl-coenzyme M Reductase and Its Nickel Corphin Coenzyme F430 in Methanogenic Archaea. In Nickel and its Surprising Impact in Nature; Sigel, A., Sigel, H., Sigel, R. K. O., Eds.; John Wiley & Sons: Chichester, 2007; pp 323-356. (8) Crabtree, R. Chem. ReV. 1995, 95, 987. (9) Van, Doorslaer, S.; Vionck, E. Phys. Chem. Chem. Phys. 2007, 9, 4620. (10) Review, in Portuguese: Nakagaki, S.; Friedermann, G. R.; Caiut, J. M. A. Quim. NoVa 2006, 29, 1003. (11) Ragsdale, S. W. J. Inorg. Biochem. 2007, 101, 1657. (12) Ghosh, A.; Wondimagegn, T.; Ryeng, H. Curr. Opin. Chem. Biol. 2001, 5, 744. (13) Wondimagegn, T.; Ghosh, A. J. Am. Chem. Soc. 2000, 122, 6375. (14) Wondimagegn, T.; Ghosh, A. J. Am. Chem. Soc. 2001, 123, 1543. (15) (a) Zimmer, M.; Crabtree, R. H. J. Am. Chem. Soc. 1990, 112, 1062. (b) Furenlid, L. R.; Renner, M. W.; Fajer, J. J. Am. Chem. Soc. 1990, 112, 8987. (c) Waditschatka, R.; Diener, E.; Eschenmoser, A. Angew. Chem., Int. Ed. Engl. 1983, 22, 631. (d) Rasetti, V.; Pfaltz, A.; Kratky, C.; Eschenmoser, A. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 16. (e) Mu¨ller, P. M.; Farooq, S.; Hardegger, B.; Salmond, W. S.; Eschenmoser, A. Angew. Chem., Int. Ed. Engl. 1973, 12, 914. (16) Mbofana, C.; Zimmer, M. Inorg. Chem. 2006, 45, 2598. (17) Todd, L. N.; Zimmer, M. Inorg. Chem. 2002, 41, 6831. (18) Wondimagegn, T.; Ghosh, A. J. Phys. Chem. B 2000, 104, 10858. (19) Shelnutt, J. A.; Song, X.-Z.; Ma, J.-G.; Jia, S.-L.; Jentzen, W.; Medforth, C. J. Chem. Soc. ReV. 1998, 27, 31.

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