Geometric and electronic structures of the Ni(I) and methyl-Ni(III) intermediates of methyl-coenzyme M reductase

Abstract

Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in the formation of biological methane from methyl-coenzyme M (Me-SCoM) and coenzyme B (CoBSH). The active site in MCR contains a Ni-F(430) cofactor, which can exist in different oxidation states. The catalytic mechanism of methane formation has remained elusive despite intense spectroscopic and theoretical investigations. On the basis of spectroscopic and crystallographic data, the first step of the mechanism is proposed to involve a nucleophilic attack of the Ni(I) active state (MCR(red1)) on Me-SCoM to form a Ni(III)-methyl intermediate, while computational studies indicate that the first step involves the attack of Ni(I) on the sulfur of Me-SCoM, forming a CH(3)(*) radical and a Ni(II)-thiolate species. In this study, a combination of Ni K-edge X-ray absorption spectroscopic (XAS) studies and density functional theory (DFT) calculations have been performed on the Ni(I) (MCR(red1)), Ni(II) (MCR(red1-silent)), and Ni(III)-methyl (MCR(Me)) states of MCR to elucidate the geometric and electronic structures of the different redox states. Ni K-edge EXAFS data are used to reveal a five-coordinate active site with an open upper axial coordination site in MCR(red1). Ni K-pre-edge and EXAFS data and time-dependent DFT calculations unambiguously demonstrate the presence of a long Ni-C bond ( approximately 2.04 A) in the Ni(III)-methyl state of MCR. The formation and stability of this species support mechanism I, and the Ni-C bond length suggests a homolytic cleavage of the Ni(III)-methyl bond in the subsequent catalytic step. The XAS data provide insight into the role of the unique F(430) cofactor in tuning the stability of the different redox states of MCR.

Figure 1

Schematic diagram showing the proposed structures upon conversion of the active MCR_red1 to MCR_{red1−silent} and MCR_Me. The Ni^III−PS state is analogous to the Ni^III−methyl state and is formed by the reaction of MCR_red1 with bromopropane sulfonate.

Figure 2

(A) Normalized Ni K-edge XAS spectra of MCR_{red1−silent} (red), MCR_red1 (green), and MCR_Me (blue). The inset shows the expanded second-derivative spectrum. (B) First-derivative spectrum showing the edge inflection points.

Figure 3

Comparison of the k³-weighted Ni K-edge EXAFS for MCR_{red1−silent} (red), MCR_red1 (green), and MCR_Me (blue) and their corresponding Fourier transforms.

Figure 4

k³-weighted Ni K-edge EXAFS data (inset) and their corresponding Fourier transforms (FT) for (A) MCR_{red1−silent} [data (gray) and fit (red)], MCR_red1 [data (gray) and fit (green)], and MCR_Me [data (gray) and fit (blue)].

Figure 5

(A) Comparison of the Ni K-pre-edge XAS data (—) with the TD-DFT calculated spectra (---): MCR_{red1−silent} (red), MCR_red1 (green), MCR_Me (blue), and MCR_Me−NA (gray).

Figure 6

Schematic diagram of the predicted active site structures of MCR_{red1−silent}, MCR_red1, and MCR_Me based on Ni K-edge EXAFS and Ni K-pre-edge analysis. Relevant first-shell bond distances have been included. Some atoms of the F₄₃₀ cofactor have been omitted for the sake of clarity. The lower-axial bond distance in MCR_Me is ill-determined by EXAFS. The dashed Ni−O bond in MCR_red1 and MCR_Me indicates a larger than normal EXAFS uncertainty. Asterisks indicate the average Ni−C bond distance, based on TD-DFT calculations and EXAFS data analysis.