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Oxidation of methane by a biological dicopper centre

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.

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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.

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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)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  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)

    CAS  Article  Google Scholar 

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

    CAS  Article  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)

    ADS  CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  Google Scholar 

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

    CAS  Article  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)

    CAS  Article  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)

    ADS  CAS  Article  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)

    CAS  Article  Google Scholar 

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

    CAS  Article  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)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  32. George, G. N., George, S. J. & Pickering, I. J. EXAFSPAKhttp://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)

    ADS  CAS  Article  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)

    CAS  Article  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.

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

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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

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