Oxidation of methane by a biological dicopper centre

Journal name:
Date published:
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


  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


  1. Hermans, I., Spier, E. S., Neuenschwander, U., Turra, N. & Baiker, A. Selective oxidation catalysis: opportunities and challenges. Top. Catal. 52, 11621174 (2009)
  2. Arakawa, H. et al. Catalysis research of relevance to carbon management: progress, challenges, and opportunities. Chem. Rev. 101, 953996 (2001)
  3. Que, L. & Tolman, W. B. Biologically inspired oxidation catalysis. Nature 455, 333340 (2008)
  4. Merkx, M. et al. Dioxygen activation and methane hydroxylation by soluble methane monooxygenase: a tale of two irons and three proteins. Angew. Chem. Int. Ed. 40, 27822807 (2001)
  5. Hakemian, A. S. & Rosenzweig, A. C. The biochemistry of methane oxidation. Annu. Rev. Biochem. 76, 223241 (2007)
  6. Lieberman, R. L. & Rosenzweig, A. C. Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane. Nature 434, 177182 (2005)
  7. Balasubramanian, R. & Rosenzweig, A. C. Structural and mechanistic insights into methane oxidation by particulate methane monooxygenase. Acc. Chem. Res. 40, 573580 (2007)
  8. Lieberman, R. L. et al. Characterization of the particulate methane monooxygenase metal centers in multiple redox states by X-ray absorption spectroscopy. Inorg. Chem. 45, 83728381 (2006)
  9. Lieberman, R. L. et al. Purified particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a copper-containing cluster. Proc. Natl Acad. Sci. USA 100, 38203825 (2003)
  10. Hakemian, A. S. et al. The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b. Biochemistry 47, 67936801 (2008)
  11. Rosenzweig, A. C. The metal centres of particulate methane monooxygenase. Biochem. Soc. Trans. 36, 11341137 (2008)
  12. Chan, S. I. & Yu, S. S. F. Controlled oxidation of hydrocarbons by the membrane-bound methane monooxygenase: the case for a tricopper cluster. Acc. Chem. Res. 41, 969979 (2008)
  13. Chen, K. H.-C. et al. The copper clusters in the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath). J. Chin. Chem. Soc. 51, 10811098 (2004)
  14. Nguyen, H.-H. T. et al. X-ray absorption and EPR studies on the copper ions associated with the particulate methane monooxygenase from Methylococcus capsulatus (Bath). Cu(I) ions and their implications. J. Am. Chem. Soc. 118, 1276612776 (1996)
  15. Chan, S. I. et al. Redox potentiometry studies of particulate methane monooxygenase: support for a trinuclear copper cluster active site. Angew. Chem. Int. Ed. 46, 19921994 (2007)
  16. Yu, S. S. F. et al. The C-terminal aqueous-exposed domain of the 45 kDa subunit of the particulate methane monooxygenase in Methylococcus capsulatus (Bath) is a Cu(I) sponge. Biochemistry 46, 1376213774 (2007)
  17. Martinho, M. et al. Mössbauer studies of the membrane-associated methane monooxygenase from Methylococcus capsulatus Bath: evidence for a diiron center. J. Am. Chem. Soc. 129, 1578315785 (2007)
  18. Choi, D. W. et al. The membrane-associated methane monooxygenase pMMO and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath. J. Bacteriol. 185, 57555764 (2003)
  19. Kitmitto, A., Myronova, N., Basu, P. & Dalton, H. Characterization and structural analysis of an active particulate methane monooxygenase trimer from Methylococcus capsulatus (Bath). Biochemistry 44, 1095410965 (2005)
  20. Yu, S. S.-F. et al. Production of high-quality particulate methane monooxygenase in high yields from Methylococcus capsulatus (Bath) with a hollow-fiber membrane bioreactor. J. Bacteriol. 185, 59155924 (2003)
  21. Miyaji, A., Suzuki, M., Baba, T., Kamachi, T. & Okura, I. Hydrogen peroxide as an effecter on the inactivation of particulate methane monooxygenase under aerobic conditions. J. Mol. Catal. B 57, 211215 (2009)
  22. Kau, L.-S., Spira-Solomon, D. J., Penner-Hahn, J. E., Hodgson, K. O. & Solomon, E. I. X-ray absorption edge determination of the oxidation state and coordination number of copper. Application to the type 3 site in Rhus vernicifera laccase and its reaction with oxygen. J. Am. Chem. Soc. 109, 64336442 (1987)
  23. Yatsunyk, L. A. & Rosenzweig, A. C. Copper(I) binding and transfer by the N-terminus of the Wilson disease protein. J. Biol. Chem. 282, 86228631 (2007)
  24. Prior, S. D. & Dalton, H. Acetylene as a suicide substrate and active site probe for methane monooxygenase from Methylococcus capsulatus (Bath). FEMS Microbiol. Lett. 29, 105109 (1985)
  25. Zahn, J. A. & DiSpirito, A. A. Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath). J. Bacteriol. 178, 10181029 (1996)
  26. Shiota, Y. & Yoshizawa, K. Comparison of the reactivity of bis(μ-oxo)CuIICuIII and CuIIICuIII species to methane. Inorg. Chem. 48, 838845 (2009)
  27. Woertink, J. S. et al. A [Cu2O]2+ core in Cu-ZSM-5, the active site in the oxidation of methane to methanol. Proc. Natl Acad. Sci. USA 106, 1890818913 (2009)
  28. Miller, K. W., Hammond, L. & Porter, E. G. The solubility of hydrocarbon gases in lipid bilayers. Chem. Phys. Lipids 20, 229241 (1977)
  29. Batliwala, H., Somasundaram, T., Uzgiris, E. E. & Makowski, L. Methane-induced hemolysis of human erythrocytes. Biochem. J. 307, 433438 (1995)
  30. Myronova, N., Kitmitto, A., Collins, R. F., Miyaji, A. & Dalton, H. Three-dimensional structure determination of a protein supercomplex that oxidizes methane to formaldehyde in Methylococcus capsulatus (Bath). Biochemistry 45, 1190511914 (2006)
  31. Stookey, L. L. Ferrozine - a new spectrophotometric reagent for iron. Anal. Chem. 42, 779781 (1970)
  32. George, G. N., George, S. J. & Pickering, I. J. EXAFSPAK left fencehttp://www-ssrl.slac.stanford.edu/~george/exafspak/exafs.htmright fence (2001)
  33. Ankudinov, A. L. & Rehr, J. J. Relativistic calculations of spin-dependent X-ray absorption spectra. Phys. Rev. B 56, R1712R1716 (1997)
  34. Riggs-Gelasco, P. J., Stemmler, T. L. & Penner-Hahn, J. E. XAFS of dinuclear metal sites in proteins and model compounds. Coord. Chem. Rev. 144, 245286 (1995)

Download references

Author information

  1. These authors contributed equally to this work.

    • Ramakrishnan Balasubramanian &
    • Stephen M. Smith


  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

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Information (1.9M)

    This file contains Supplementary Tables S1-S3 and Supplementary Figures S1-S5 with legends.

Additional data