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

Abstract

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.

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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.

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Acknowledgements

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.).

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Authors

Contributions

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.

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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

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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). https://doi.org/10.1038/nature14160

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