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

Abstract

We present the 1.2 Å resolution X-ray crystal structure of a Ni-methyl species that is a proposed catalytic intermediate in methyl-coenzyme M reductase (MCR); the enzyme that catalyzes the biological formation of methane. The methyl group is situated 2.1 Å proximal of the Ni-atom of the MCR coenzyme F₄₃₀. A rearrangement of the substrate channel has been posited to bring together substrate species, but Ni(III)-methyl formation alone does not lead to any observable structural changes in the channel.

Methane has potential as an alternative fuel, but is also a potent greenhouse gas with surplus methanogenesis currently leading to a slow increase in methane concentrations within the atmosphere. The major source of methane produced in nature is the end product of the archaeal decomposition of organic matter in strict anaerobic environments such as lake sediments and the intestinal tract of animals.¹ In the methanogenic archaea, methyl-coenzyme M reductase (MCR) catalyzes the final and rate-limiting step in methane biogenesis; the reduction of methyl-coenzyme M (methyl-SCoM, 2-(methylthio)ethanesulfonate) by coenzyme B (CoBSH, N-7-mercaptoheptanoylthreonine phosphate) to methane and a heterodisulfide (Supporting Information, Scheme S1). The 273 kDa MCR enzyme contains two deeply buried active sites, each containing a highly reduced Ni-tetrapyrrole cofactor, named coenzyme F₄₃₀ (F₄₃₀)². The Ni atom in F₄₃₀ is redox active and has been proposed to shuttle between oxidation states +1, +2 and +3 during turnover^1,3,4 (Scheme 1.1), whilst other studies have suggested the involvement of only Ni oxidation states +1 and +2 (Supporting Information Scheme S2)^5–7. The actual mechanism remains elusive since no true catalytic intermediate of MCR has ever been observed. However, a stable MCR-Ni(III)-methyl (MCR_Me) state can be artificially produced by treating the resting active Ni(I) form of the enzyme (MCR_red1) with methyl halide (Scheme 1.2).^8,9 Treatment of MCR_Me with coenzyme M (HSCoM, demethylated methyl-SCoM) generates MCR_red1 along with the substrate methyl-SCoM, supporting this species as a possible catalytic intermediate.⁸

Scheme 1.

(1) Proposed MCR mechanism involving a Ni(III)-methyl intermediate. (2) Generation of a Ni(III)-methyl species using methyl iodide. The resting Ni(I) state is circled with a dashed line. For clarity the axial glutamine residue has been excluded

The geometric and electronic structure of MCR_Me was recently characterized in solution by X-ray absorption spectroscopy (XAS) and density functional theory (DFT).¹⁰ Here we describe the X-ray crystal structure of the protein in this mechanistically relevant state. To confirm that MCR_Me could form in crystals, a Ni K-edge XAS spectrum was obtained from a MCR_red1 crystal treated anaerobically with methyl iodide, which demonstrated MCR_Me formation in the crystal (Supporting Information Methods, Results and Figure S1).

X-ray diffraction data were collected on a MCR_Me crystal, and modeling and refinement led to final R-values are 13.2 % and 15.6 % for R_work and R_free respectively (Supporting Information Methods, Results and Table S1) The electron density map at 1.2 Å resolution contained two distinct species bound proximal to the Ni of F₄₃₀. Besides a background of bound HSCoM (added during purification to stabilize MCR) there was clear Fo-Fc difference density consistent with a Ni-coordinated methyl group (Figure 1A). To deconvolute the multiple species from within the active site anomalous data generated from the native dataset (wavelength 0.98 Å) and at two additional wavelengths, 1.51 Å and 2.29 Å, were used (Figure 1B, Supporting Information Methods, Results, Table S2 and Figure S2). The Ni-S distance refines to 2.4 Å which is in line with the previous crystal structures containing HSCoM^2,11–13 and the distance reported by XAS.¹⁰ The Ni-C distance refines to 2.07 Å and 2.10 Å in the two crystallographically independent active sites in the asymmetric unit (the distance determined by XAS for MCR_Me in solution is 2.04 Ź⁰). The substrate channel contains a mixture of coenzyme B analogue (CoB₆SH (Figure S3); a slow substrate added in crystallization) and CoBSH (co-purifies with the enzyme: Figure 2). The respective thiols of CoB₆SH and the substrate CoBSH are coincident and situated ~8.7 Å away from the Ni in F₄₃₀.¹¹ As CoBSH is a substrate, the long distance between the thiol and Ni(II) in the previous inactive MCR crystal structures has been puzzling.^2,11–13 Thus, a conformational change has been proposed that would bring the CoBSH closer to the Ni,^11,15 which is consistent with recent transient kinetic studies revealing substrate-triggered conformational changes in the catalytic resting state MCR_red1.¹⁶

Figure 1.

Ni-methyl MCR electron density. A) Stereo figure showing positive Fo − Fc electron density (green mesh, contoured at 5σ) when methyl is not included in the structural model. 2Fo − Fc electron density (contoured at 1σ) is shown as blue mesh. B) Anomalous electron density map from X-ray diffraction data collected at wavelength 2.29 Å (orange mesh, contoured at 5σ) (f″ (electrons): sulfur 1.14; nickel 1.03). Coenzymes are shown in stick colored by atom (carbon: CoB₆SH, pale yellow; CoBSH, pale green; HSCoM, light grey (faded); coenzyme F₄₃₀, dark grey with the Ni atom shown as green sphere). Iodide as a purple sphere and a water molecule as a red sphere. Protein is shown as cartoon.

Figure 2.

Final structural model and electron density. The final model contains a 60:40 mixture of coenzyme B₆ (CoB₆SH) and coenzyme B (CoBSH), 50% coenzyme M (HSCoM), 50 % methyl and 35% iodide. Coenzymes are shown in stick colored by atom (carbon: CoB₆SH, pale yellow; CoBSH, pale green; HSCoM, light grey (faded); coenzyme F₄₃₀, dark grey with the Ni atom shown as green sphere). Methyl is displayed as a grey sphere, iodide as a purple sphere and a water molecule as a red sphere. Protein is shown as cartoon. 2Fo − Fc electron density (contoured at 1σ) is shown as blue mesh.

Our data suggest that if Ni(III)-methyl is an intermediate in methane formation, its presence alone does not affect the architecture of the MCR substrate channel, which appears unchanged compared to the structure of the channel in the Ni(II) structures. The release of methane involves a protonation step, which has been postulated to either be provided by CoBSH via a CoM species (Scheme 1.1), or possibly via a water molecule (Figure 2), two Tyr residues² (Tyrα333 and Tyrβ367, whose hydroxyls point towards the Ni) or the C-ring nitrogen of F₄₃₀³. Since no catalytic intermediates of MCR have been observed thus far by fast spectroscopic techniques, any Ni(III)-methyl catalytic intermediate formed from the native substrates must be fleeting during turnover. Recently transient kinetic methods using the natural substrate, methyl-SCoM, and the slow substrate CoB₆SH, enabled observation of a short-lived Ni(III)-alkyl intermediate and its subsequent decay at catalytically competent rates.¹⁶ In contrast, when Ni(III)-methyl is artificially generated using methyl iodide, as in this study, it is relatively stable enabling spectroscopic and biochemical characterization.^8,9

Interestingly, MCR is also implicated in “reverse” methanogenesis in the anaerobic oxidation of methane within microbial mats found at methane seeps in the deep ocean.^17,18 The dissociation energy of the C-H bond (+439 kJ/mol) is larger than in any other organic compound except benzene. Thauer and Shima have proposed that in “reverse” methanogenesis only the strong electrophile F₄₃₀-Ni(III) of MCR is capable of breaking the C-H bond in a modified “reversed” version of the Ni(III)-methyl mechanism proposed for methanogenesis (Scheme 1.1).¹⁹ The recent evidence of a Ni-(σ-alkane) intermediate in the reaction of MCR is consistent with this proposal.²⁰

The crystal structure of MCR_Me is the first structure resembling a postulated intermediate in one of the proposed mechanisms of MCR. The high resolution of the X-ray diffraction data, coupled with anomalous diffraction at several wavelengths, has enabled us to extract the relevant Ni-methyl structure from confounding species. The active Ni(I) enzyme, its complex with methyl-SCoM, substrate channel conformational changes and true catalytic intermediates trapped during turnover still wait to be structurally defined.

Abbreviations

MCR

Methyl-coenzyme M reductase

F430

coenzyme F430

methyl-SCoM

methyl-coenzyme M

CoBSH

coenzyme B

HSCoM

coenzyme M

CoB₆SH

coenzyme B analogue