Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Oxidation of methane by a biological dicopper centre

Nature volume 465, pages 115–119 (2010)Cite this article

Subjects

Abstract

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 (104 kcal mol-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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure of the M. capsulatus (Bath) pMMO protomer.
Figure 2: Metal analysis.
Figure 3: Restoration of activity to apo-pMMO by the addition of copper.
Figure 4: Copper EXAFS data and simulations for pMMO and spmoB variants.
Figure 5: Catalytic activity of spmoB proteins.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Hermans, I., Spier, E. S., Neuenschwander, U., Turra, N. & Baiker, A. Selective oxidation catalysis: opportunities and challenges. Top. Catal. 52, 1162–1174 (2009)

    Article  CAS  Google Scholar 

  2. Arakawa, H. et al. Catalysis research of relevance to carbon management: progress, challenges, and opportunities. Chem. Rev. 101, 953–996 (2001)

    Article  CAS  Google Scholar 

  3. Que, L. & Tolman, W. B. Biologically inspired oxidation catalysis. Nature 455, 333–340 (2008)

    Article  ADS  CAS  Google Scholar 

  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, 2782–2807 (2001)

    Article  CAS  Google Scholar 

  5. Hakemian, A. S. & Rosenzweig, A. C. The biochemistry of methane oxidation. Annu. Rev. Biochem. 76, 223–241 (2007)

    Article  CAS  Google Scholar 

  6. Lieberman, R. L. & Rosenzweig, A. C. Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane. Nature 434, 177–182 (2005)

    Article  ADS  CAS  Google Scholar 

  7. Balasubramanian, R. & Rosenzweig, A. C. Structural and mechanistic insights into methane oxidation by particulate methane monooxygenase. Acc. Chem. Res. 40, 573–580 (2007)

    Article  CAS  Google Scholar 

  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, 8372–8381 (2006)

    Article  CAS  Google Scholar 

  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, 3820–3825 (2003)

    Article  ADS  CAS  Google Scholar 

  10. Hakemian, A. S. et al. The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b. Biochemistry 47, 6793–6801 (2008)

    Article  CAS  Google Scholar 

  11. Rosenzweig, A. C. The metal centres of particulate methane monooxygenase. Biochem. Soc. Trans. 36, 1134–1137 (2008)

    Article  CAS  Google Scholar 

  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, 969–979 (2008)

    Article  CAS  Google Scholar 

  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, 1081–1098 (2004)

    Article  CAS  Google Scholar 

  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, 12766–12776 (1996)

    Article  CAS  Google Scholar 

  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, 1992–1994 (2007)

    Article  CAS  Google Scholar 

  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, 13762–13774 (2007)

    Article  CAS  Google Scholar 

  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, 15783–15785 (2007)

    Article  CAS  Google Scholar 

  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, 5755–5764 (2003)

    Article  CAS  Google Scholar 

  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, 10954–10965 (2005)

    Article  CAS  Google Scholar 

  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, 5915–5924 (2003)

    Article  CAS  Google Scholar 

  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, 211–215 (2009)

    Article  CAS  Google Scholar 

  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, 6433–6442 (1987)

    Article  CAS  Google Scholar 

  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, 8622–8631 (2007)

    Article  CAS  Google Scholar 

  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, 105–109 (1985)

    Article  CAS  Google Scholar 

  25. Zahn, J. A. & DiSpirito, A. A. Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath). J. Bacteriol. 178, 1018–1029 (1996)

    Article  CAS  Google Scholar 

  26. Shiota, Y. & Yoshizawa, K. Comparison of the reactivity of bis(μ-oxo)CuIICuIII and CuIIICuIII species to methane. Inorg. Chem. 48, 838–845 (2009)

    Article  CAS  Google Scholar 

  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, 18908–18913 (2009)

    Article  ADS  CAS  Google Scholar 

  28. Miller, K. W., Hammond, L. & Porter, E. G. The solubility of hydrocarbon gases in lipid bilayers. Chem. Phys. Lipids 20, 229–241 (1977)

    Article  CAS  Google Scholar 

  29. Batliwala, H., Somasundaram, T., Uzgiris, E. E. & Makowski, L. Methane-induced hemolysis of human erythrocytes. Biochem. J. 307, 433–438 (1995)

    Article  CAS  Google Scholar 

  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, 11905–11914 (2006)

    Article  CAS  Google Scholar 

  31. Stookey, L. L. Ferrozine - a new spectrophotometric reagent for iron. Anal. Chem. 42, 779–781 (1970)

    Article  CAS  Google Scholar 

  32. George, G. N., George, S. J. & Pickering, I. J. EXAFSPAK 〈http://www-ssrl.slac.stanford.edu/~george/exafspak/exafs.htm〉 (2001)

    Google Scholar 

  33. Ankudinov, A. L. & Rehr, J. J. Relativistic calculations of spin-dependent X-ray absorption spectra. Phys. Rev. B 56, R1712–R1716 (1997)

    Article  ADS  CAS  Google Scholar 

  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, 245–286 (1995)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by US National Institutes of Health (NIH) grants GM070473 (A.C.R.) and DK068139 (T.L.S.). Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource (SSRL). The SSRL is a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the NIH, National Center for Research Resources, Biomedical Technology Program.

Author Contributions R.B., S.M.S., S.R. and L.A.Y. performed experiments. R.B., S.M.S., S.R., T.L.S. and A.C.R. contributed to experimental design, data analysis and manuscript preparation.

Author information

Author notes

  1. Ramakrishnan Balasubramanian and Stephen M. Smith: These authors contributed equally to this work.

Authors and 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

Authors
  1. Ramakrishnan Balasubramanian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stephen M. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Swati Rawat
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Liliya A. Yatsunyk
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Timothy L. Stemmler
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Amy C. Rosenzweig
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Timothy L. Stemmler or Amy C. Rosenzweig.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables S1-S3 and Supplementary Figures S1-S5 with legends. (PDF 1970 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Balasubramanian, R., Smith, S., Rawat, S. et al. Oxidation of methane by a biological dicopper centre. Nature 465, 115–119 (2010). https://doi.org/10.1038/nature08992

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08992

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing