Skip to main content

Thank you for visiting 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.

Structure of the key species in the enzymatic oxidation of methane to methanol


Methane monooxygenase (MMO) catalyses the O2-dependent conversion of methane to methanol in methanotrophic bacteria, thereby preventing the atmospheric egress of approximately one billion tons of this potent greenhouse gas annually. The key reaction cycle intermediate of the soluble form of MMO (sMMO) is termed compound Q (Q). Q contains a unique dinuclear FeIV cluster that reacts with methane to break an exceptionally strong 105 kcal mol−1 C-H bond and insert one oxygen atom1,2. No other biological oxidant, except that found in the particulate form of MMO, is capable of such catalysis. The structure of Q remains controversial despite numerous spectroscopic, computational and synthetic model studies2,3,4,5,6,7. A definitive structural assignment can be made from resonance Raman vibrational spectroscopy but, despite efforts over the past two decades, no vibrational spectrum of Q has yet been obtained. Here we report the core structures of Q and the following product complex, compound T, using time-resolved resonance Raman spectroscopy (TR3). TR3 permits fingerprinting of intermediates by their unique vibrational signatures through extended signal averaging for short-lived species. We report unambiguous evidence that Q possesses a bis-μ-oxo diamond core structure and show that both bridging oxygens originate from O2. This observation strongly supports a homolytic mechanism for O-O bond cleavage. We also show that T retains a single oxygen atom from O2 as a bridging ligand, while the other oxygen atom is incorporated into the product8. Capture of the extreme oxidizing potential of Q is of great contemporary interest for bioremediation and the development of synthetic approaches to methane-based alternative fuels and chemical industry feedstocks. Insight into the formation and reactivity of Q from the structure reported here is an important step towards harnessing this potential.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Reaction of sMMO with O2.
Figure 3: Formation of compound Q and its reaction with methane.
Figure 2: Fingerprinting cluster structure using 16O18O mixed oxygen isotope.


  1. Lee, S. K., Nesheim, J. C. & Lipscomb, J. D. Transient intermediates of the methane monooxygenase catalytic cycle. J. Biol. Chem. 268, 21569–21577 (1993)

    CAS  PubMed  Google Scholar 

  2. Lee, S. K., Fox, B. G., Froland, W. A., Lipscomb, J. D. & Münck, E. A transient intermediate of the methane monooxygenase catalytic cycle containing a FeIVFeIV cluster. J. Am. Chem. Soc. 115, 6450–6451 (1993)

    CAS  Article  Google Scholar 

  3. Shu, L. et al. An FeIV2O2 diamond core structure for the key intermediate Q of methane monooxygenase. Science 275, 515–518 (1997)

    CAS  Article  PubMed  Google Scholar 

  4. Dunietz, B. D. et al. Large scale ab initio quantum chemical calculation of the intermediates in the soluble methane monooxygenase catalytic cycle. J. Am. Chem. Soc. 122, 2828–2839 (2000)

    CAS  Article  Google Scholar 

  5. Han, W. G. & Noodleman, L. Structural model studies for the high-valent intermediate Q of methane monooxygenase from broken-symmetry density functional calculations. Inorganica Chim. Acta 361, 973–986 (2008)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Siegbahn, P. E. M. O−O bond cleavage and alkane hydroxylation in methane monooxygenase. J. Biol. Inorg. Chem. 6, 27–45 (2001)

    CAS  Article  PubMed  Google Scholar 

  7. Xue, G. et al. A synthetic precedent for the [FeIV2(µ-O)2] diamond core proposed for methane monooxygenase intermediate Q. Proc. Natl Acad. Sci. USA 104, 20713–20718 (2007)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  8. Nesheim, J. C. & Lipscomb, J. D. Large isotope effects in methane oxidation catalyzed by methane monooxygenase: evidence for C−H bond cleavage in a reaction cycle intermediate. Biochemistry 35, 10240–10247 (1996)

    CAS  Article  PubMed  Google Scholar 

  9. Liu, K. E. et al. Spectroscopic detection of intermediates in the reaction of dioxygen with the reduced methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath). J. Am. Chem. Soc. 116, 7465–7466 (1994)

    CAS  Article  Google Scholar 

  10. Tinberg, C. E. & Lippard, S. J. Revisiting the mechanism of dioxygen activation in soluble methane monooxygenase from M. capsulatus (Bath): evidence for a multi-step, proton-dependent reaction pathway. Biochemistry 48, 12145–12158 (2009)

    CAS  Article  PubMed  Google Scholar 

  11. Banerjee, R., Meier, K. K., Münck, E. & Lipscomb, J. D. Intermediate P* from soluble methane monooxygenase contains a diferrous cluster. Biochemistry 52, 4331–4342 (2013)

    CAS  Article  PubMed  Google Scholar 

  12. Grzyska, P. K., Appelman, E. H., Hausinger, R. P. & Proshlyakov, D. A. Insight into the mechanism of an iron dioxygenase by resolution of steps following the FeIV = O species. Proc. Natl Acad. Sci. USA 107, 3982–3987 (2010)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  13. Zheng, H. & Lipscomb, J. D. Regulation of methane monooxygenase catalysis based on size exclusion and quantum tunneling. Biochemistry 45, 1685–1692 (2006)

    CAS  Article  PubMed  Google Scholar 

  14. Hohenberger, J., Kallol, R. & Meyer, K. K. The biology and chemistry of high-valent iron–oxo and iron–nitrido complexes. Nat. Commun. 3, 720 (2012)

    Article  ADS  PubMed  Google Scholar 

  15. Vu, V. V. et al. Human deoxyhypusine hydroxylase, an enzyme involved in regulating cell growth, activates O2 with a nonheme diiron center. Proc. Natl Acad. Sci. USA 106, 14814–14819 (2009)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  16. Shiemke, A. K., Loehr, T. M. & Sanders-Loehr, J. Resonance Raman study of oxyhemerythrin and hydroxhemerythrin: evidence for hydrogen bonding of ligands to the Fe-O-Fe center. J. Am. Chem. Soc. 108, 2437–2443 (1986)

    CAS  Article  PubMed  Google Scholar 

  17. Stone, K. L., Behan, R. K. & Green, M. T. Resonance Raman spectroscopy of chloroperoxidase compound II provides direct evidence for the existence of an iron(IV)-hydroxide. Proc. Natl Acad. Sci. USA 103, 12307–12310 (2006)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  18. Momenteau, M. & Reed, C. A. Synthetic heme dioxygen complexes. Chem. Rev. 94, 659–698 (1994)

    CAS  Article  Google Scholar 

  19. Wilkinson, E. C. et al. Raman signature of the Fe2O2 “diamond” core. J. Am. Chem. Soc. 120, 955–962 (1998)

    CAS  Article  Google Scholar 

  20. Tinberg, C. E. & Lippard, S. J. Dioxygen activation in soluble methane monooxygenase. Acc. Chem. Res. 44, 280–288 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Poulos, T. L. Heme enzyme structure and function. Chem. Rev. 114, 3919–3962 (2014)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Tolman, W. B. Making and breaking the dioxygen O-O bond: new insights from studies of synthetic copper complexes. Acc. Chem. Res. 30, 227–237 (1997)

    CAS  Article  Google Scholar 

  23. Collman, J. P., Dey, A., Yang, Y., Ghosh, S. & Decreau, R. A. O2 reduction by a functional heme/nonheme bis-iron NOR model complex. Proc. Natl Acad. Sci. USA 106, 10528–10533 (2009)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  24. Xue, G., De Hont, R., Münck, E. & Que, L., Jr Million-fold activation of the [Fe2(µ-O)2] diamond core for C-H bond cleavage. Nat. Chem. 2, 400–405 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Zheng, H., Zang, Y., Dong, Y., Young, V. G., Jr & Que, L., Jr Complexes with FeIII2(µ-O)(µ-OH), FeIII2(µ-O)2, and [FeIII3(µ2-O)3] cores: Structures, spectroscopy, and core interconversions. J. Am. Chem. Soc. 121, 2226–2235 (1999)

    CAS  Article  Google Scholar 

  26. Sjöberg, B.-M., Loehr, T. M. & Sanders-Loehr, J. Raman spectral evidence for a μ-oxo bridge in the binuclear iron center of ribonucleotide reductase. Biochemistry 21, 96–102 (1982)

    Article  PubMed  Google Scholar 

  27. Fox, B. G., Shanklin, J., Ai, J., Loehr, T. M. & Sanders-Loehr, J. Resonance Raman evidence for an Fe-O-Fe center in stearoyl-ACP desaturase. Primary sequence identity with other diiron-oxo proteins. Biochemistry 33, 12776–12786 (1994)

    CAS  Article  PubMed  Google Scholar 

  28. Carter, E. L., Proshlyakov, D. A. & Hausinger, R. P. Apoprotein isolation and activation, and vibrational structure of the Helicobacter mustelae iron urease. J. Inorg. Biochem. 111, 195–202 (2012)

    CAS  Article  PubMed  Google Scholar 

  29. Sitter, A. J., Shifflett, J. R. & Terner, J. Resonance Raman spectroscopic evidence for heme iron-hydroxide ligation in peroxidase alkaline forms. J. Biol. Chem. 263, 13032–13038 (1988)

    CAS  PubMed  Google Scholar 

  30. Zhang, J. & Lipscomb, J. D. Role of the C-terminal region of the B component of Methylosinus trichosporium OB3b methane monooxygenase in the regulation of oxygen activation. Biochemistry 45, 1459–1469 (2006)

    CAS  Article  PubMed  Google Scholar 

  31. Fox, B. G., Froland, W. A., Dege, J. E. & Lipscomb, J. D. Methane monooxygenase from Methylosinus trichosporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph. J. Biol. Chem. 264, 10023–10033 (1989)

    CAS  PubMed  Google Scholar 

  32. Brazeau, B. J. & Lipscomb, J. D. Kinetics and activation thermodynamics of methane monooxygenase compound Q and reaction with substrates. Biochemistry 39, 13503–13515 (2000)

    CAS  Article  PubMed  Google Scholar 

  33. Lee, S. K. & Lipscomb, J. D. Oxygen activation catalyzed by methane monooxygenase hydroxylase component: Proton delivery during the O-O bond cleavage steps. Biochemistry 38, 4423–4432 (1999)

    CAS  Article  PubMed  Google Scholar 

  34. Rosenzweig, A. C., Nordlund, P., Takahara, P. M., Frederick, C. A. & Lippard, S. J. Geometry of the soluble methane monooxygenase catalytic diiron center in two oxidation states. Chem. Biol. 2, 409–418 (1995)

    CAS  Article  PubMed  Google Scholar 

  35. Srnec, M. et al. Structural and spectroscopic properties of the peroxodiferric intermediate of Ricinus communis soluble Δ9 desaturase. Inorg. Chem. 51, 2806–2820 (2012)

    CAS  Article  PubMed  Google Scholar 

  36. Jensen, K. P., Bell, C. B., III, Clay, M. D. & Solomon, E. I. Peroxo-type intermediates in class I ribonucleotide reductase and related binuclear non-heme iron enzymes. J. Am. Chem. Soc. 131, 12155–12171 (2009)

    CAS  Article  PubMed  Google Scholar 

  37. Han, W. G. & Noodleman, L. Structural model studies for the peroxo intermediate P and the reaction pathway from P→Q of methane monooxygenase using broken-symmetry density functional calculations. Inorg. Chem. 47, 2975–2986 (2008)

    CAS  Article  PubMed  Google Scholar 

  38. England, J. et al. A synthetic high-spin oxoiron(IV) complex: Generation, spectroscopic characterization, and reactivity. Angew. Chem. Int. Ed. 48, 3622–3626 (2009)

    CAS  Article  Google Scholar 

  39. Rohde, J.-U. et al. Crystallographic and spectroscopic characterization of a nonheme Fe(IV) = O complex. Science 299, 1037–1039 (2003)

    CAS  Article  ADS  PubMed  Google Scholar 

  40. Broadwater, J. A., Ai, J., Loehr, T. M., Sanders-Loehr, J. & Fox, B. G. Peroxodiferric intermediate of stearoyl-acyl carrier protein Δ9 desaturase: Oxidase reactivity during single turnover and implications for the mechanism of desaturation. Biochemistry 37, 14664–14671 (1998)

    CAS  Article  PubMed  Google Scholar 

  41. Moenne-Loccoz, P. et al. The ferroxidase reaction of ferritin reveals a diferric μ-1,2 bridging peroxide intermediate in common with other O2-activating non-heme diiron proteins. Biochemistry 38, 5290–5295 (1999)

    CAS  Article  PubMed  Google Scholar 

Download references


We thank G. T. Babcock (deceased), S.-K. Lee and J. C. Nesheim (deceased) for initial studies that led to this project and E. Bergeron for technical assistance. This work was supported by the NIH grants GM40466 and GM100943 (to J.D.L.) and grant GM096132 (to D.A.P.).

Author information

Authors and Affiliations



R.B., Y.P. and D.A.P. developed the enhanced instrument and performed the experiments, D.A.P. analysed the data, and R.B., D.A.P. and J.D.L. designed the experiments and wrote the manuscript.

Corresponding authors

Correspondence to John D. Lipscomb or Denis A. Proshlyakov.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Speciation plots of the sMMO reaction with O2.

Plots were computed using known rate constants of the catalytic steps1,8,32,33 for the following conditions. a, No added substrate, b, presence of 0.45 mM CH4, c, presence of 3.5 mM furan and d, presence of 0.45 mM CD4 at pH 7.0, 4 °C. Rate constants used in simulation of individual conditions are shown for each step. All rate constants are first order (s−1) except for the Q to T step, which is first order in both Q and substrate (M−1 s−1) but is given as a pseudo first order constant for the current substrate concentration. The rate constant for the formation of intermediate O (oxygen binding) is unknown, but it is assumed to be fast based upon typical rates for metalloenzymes of the MMO type. It is irreversible because the rate constant of the next step (O → P*) is independent of O2 concentration.

Extended Data Figure 2 Absolute TR3 spectra of the sMMO reaction with O2.

Top, reaction using 16O2-containing buffer (blue), 18O2-containing buffer (red) or buffer background in the absence of MMOH/MMOB (black). Middle, 16O2 – 18O2 difference spectra of the MMOHred/MMOB reaction with O2 at pH 7.0, 4 °C and Δt ≈ 3.0 s. Bottom, 16O2 – 18O2 difference spectra of the oxygenated buffers in the absence of MMOH and MMOB proteins. Intensities of sMMO spectra were normalized to protein vibration. In the absence of protein, relative intensity was normalized using buffer vibrations.

Extended Data Figure 3 Power dependence of TR3 spectra of sMMO.

16O2 – 18O2 difference spectra obtained using 65 mW (i) and 15 mW (ii) excitation laser power show the same normalized intensity of oxygen vibrations in Q, indicating that no detectable photodecomposition is taking place under current conditions.

Extended Data Figure 4 A comparison of the electronic absorption spectra of compound T (red trace) and MMOHred (blue trace).

T exhibits an absorption band in the near-ultraviolet region, giving rise to its resonance Raman enhancement. Single wavelength time courses of the reaction of 25 μM MMOHred/MMOB with a 450 μM solution of CH4 and 450 μM O2 at 4 °C, pH 7 were recorded throughout the visible region (concentrations after mixing). The absorbance at each wavelength at the time of maximal T formation given by the speciation plot shown in Extended Data Fig. 1b was extracted and used to make the red trace shown.

Extended Data Figure 5 Potential O-O bond cleavage mechanisms in the dinuclear centre of MMO.

The most divergent mechanisms are shown along with expected isotopic composition of oxygen derived from O2 (red) and solvent (black). All mechanisms are triggered by proton-dependent rearrangement of P10,33. The monodentate carboxylate bridge (E243) found in the diferrous enzyme34 is likely to maintain this position in P, but return to the non-bridging position in Q, as found in the resting enzyme, to accommodate the diamond core structure. The catalytic base B, which mediates proton dependency, has not been definitively identified. Based on structural similarity to other di-iron O2-activating enzymes and DFT computations for P-analogues in those systems11,35,36, we have proposed11 that E114 (a ligand to solvent-coordinated iron in P), is this base. Other ligands not directly involved in cleavage are omitted for clarity (see Fig. 3). Equal intensities of Q-16O2 and Q-18O2 modes, and the absence of Q-16O18O mode in Fig. 2c, (i) argue against isotope scrambling in Q formation. This and all other experimental results reported to date are in full accord with the nominally concerted homolytic cleavage mechanism. We postulate that the loss of E243 bridge facilitates the conversion of cis-μ-peroxo adduct in P to the trans-μ-peroxo conformation and the ensuing O-O bond cleavage (a) to form the diamond core structure detected here. This transition is supported by DFT computations37. In contrast, the stepwise, end-on heterolytic cleavage mechanism (c) (analogous to formation of compound I in cytochrome P450) leads to the mixed isotope cluster in Q-18O2 and can be ruled out. Heterolytic cleavage of trans-μ-peroxo is essentially isoelectronic to and experimentally indistinguishable from the homolytic mechanism (a). The proton-assisted heterolytic cleavage of cis-μ-peroxo bridge (b) cannot be ruled out yet, but several observations argue against it. (1) We did not observe isotope scrambling (curved green arrows), which is expected upon formation of two terminal oxygenic ligands on the same iron. While scrambling may not occur if ligands are highly stabilized, structural basis for such putative stabilization is not apparent. Scrambling may also not be observed if formation of diamond core is fast following bond cleavage, in which case mechanism (b) becomes, in essence, a stepwise, proton-assisted homolytic cleavage, also indistinguishable from (a). (2) Two iron atoms in di-ferric P and di-ferryl Q are in the same oxidation states and in indistinguishable electronic environments2,20. Such symmetry is unfavourable for O-O bond polarization and charge separation in the FeIII/FeV state during heterolytic cleavage. The deprotonated state of the peroxo bridge in P also argues against overall polarity of the site that would aid heterolytic cleavage.

Extended Data Table 1 Additional vibrational modes of metal centres relevant to Q and T

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Banerjee, R., Proshlyakov, Y., Lipscomb, J. et al. Structure of the key species in the enzymatic oxidation of methane to methanol. Nature 518, 431–434 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


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.


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