Oxidation of methane by a biological dicopper centre

Journal name:
Nature
Volume:
465,
Pages:
115–119
Date published:
DOI:
doi:10.1038/nature08992
Received
Accepted
Published online

Vast world reserves of methane gas are underutilized as a feedstock for the production of liquid fuels and chemicals owing to the lack of economical and sustainable strategies for the selective oxidation of methane to methanol1. Current processes to activate the strong C–H bond (104kcalmol-1) in methane require high temperatures, are costly and inefficient, and produce waste2. In nature, methanotrophic bacteria perform this reaction under ambient conditions using metalloenzymes called methane monooxygenases (MMOs). MMOs thus provide the optimal model for an efficient, environmentally sound catalyst3. There are two types of MMO. Soluble MMO (sMMO) is expressed by several strains of methanotroph under copper-limited conditions and oxidizes methane with a well-characterized catalytic di-iron centre4. Particulate MMO (pMMO) is an integral membrane metalloenzyme produced by all methanotrophs and is composed of three subunits, pmoA, pmoB and pmoC, arranged in a trimeric α3β3γ3 complex5. Despite 20 years of research and the availability of two crystal structures, the metal composition and location of the pMMO metal active site are not known. Here we show that pMMO activity is dependent on copper, not iron, and that the copper active site is located in the soluble domains of the pmoB subunit rather than within the membrane. Recombinant soluble fragments of pmoB (spmoB) bind copper and have propylene and methane oxidation activities. Disruption of each copper centre in spmoB by mutagenesis indicates that the active site is a dicopper centre. These findings help resolve the pMMO controversy and provide a promising new approach to developing environmentally friendly C–H oxidation catalysts.

At a glance

Figures

  1. Structure of the M. capsulatus (Bath) pMMO protomer.
    Figure 1: Structure of the M. capsulatus (Bath) pMMO protomer.

    The amino-terminal cupredoxin domain of pmoB (spmoBd1) is shown in purple, the carboxy-terminal cupredoxin domain of pmoB (spmoBd2) is shown in green and the two transmembrane helices are shown in blue. In the recombinant spmoB protein, spmoBd1 and spmoBd2 are connected by a Gly-Lys-Leu-Gly-Gly-Gly sequence linking residues 172 and 265 (indicated), rather than the two transmembrane helices. Copper ions are shown as cyan spheres and ligands are shown as ball-and-stick representations. The pmoA (faint light green) and pmoC (faint light blue) subunits are composed of transmembrane helices. The location of the zinc ion (grey sphere) has been proposed to contain a di-iron centre. A hydrophilic patch of residues, marked with an asterisk, is the site of a proposed tricopper centre. Protein Data Bank ID, 1YEW.

  2. Metal analysis.
    Figure 2: Metal analysis.

    a, Metal content of as-isolated pMMO and apo-pMMO prepared by cyanide treatment. Metal content is expressed per 100kDa pMMO protomer, with copper in blue and iron in red. b, Copper content of refolded spmoB variants. Reported values and errors represent the average and standard deviation of at least four independent measurements for pMMO samples and at least six independent refolding experiments for each spmoB variant.

  3. Restoration of activity to apo-pMMO by the addition of copper.
    Figure 3: Restoration of activity to apo-pMMO by the addition of copper.

    Copper equivalents added are expressed per 100kDa pMMO protomer. Representative titrations are shown. Addition of 2–3equiv. of copper restored ~70% of the propylene epoxidation activity (a) and ~90% of the methane oxidation activity (b). Reported values represent the average and standard deviation of at least two measurements.

  4. Copper EXAFS data and simulations for pMMO and spmoB variants.
    Figure 4: Copper EXAFS data and simulations for pMMO and spmoB variants.

    Raw k3-weighted EXAFS data and phase-shifted Fourier transforms are shown for as-isolated pMMO (a, b), copper reconstituted pMMO (c, d), spmoB (e, f), spmoB_H48N (g, h), and spmoB_H137,139A (i, j). Raw unfiltered data are shown in black and best-fit simulations are shown in grey. χ, EXAFS region of the XAS spectrum; Δ, apparent shift in Fourier transform displayed bond distance (by ~-0.5Å) due to a phase shift during calculation of the transform; k, photoelectron wavevector; R, metal–ligand bond length.

  5. Catalytic activity of spmoB proteins.
    Figure 5: Catalytic activity of spmoB proteins.

    a, Epoxidation of propylene to propylene oxide expressed as a percentage of the activity of as-isolated, membrane-bound M. capsulatus (Bath) pMMO. b, Oxidation of methane to methanol expressed as a percentage of the activity of as-isolated, membrane-bound M. capsulatus (Bath) pMMO. All values are the average of at least two independent refolding preparations, with error bars representing standard deviations. The activity of each spmoB protein was compared with the activity of membrane-bound pMMO measured under the same experimental conditions.

Accession codes

Primary accessions

Protein Data Bank

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Author information

  1. These authors contributed equally to this work.

    • Ramakrishnan Balasubramanian &
    • Stephen M. Smith

Affiliations

  1. Department of Biochemistry, Molecular Biology and Cell Biology,

    • Ramakrishnan Balasubramanian,
    • Stephen M. Smith,
    • Liliya A. Yatsunyk &
    • Amy C. Rosenzweig
  2. Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA

    • Ramakrishnan Balasubramanian,
    • Stephen M. Smith,
    • Liliya A. Yatsunyk &
    • Amy C. Rosenzweig
  3. Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA

    • Swati Rawat &
    • Timothy L. Stemmler

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The authors declare no competing financial interests.

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