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Control of substrate access to the active site in methane monooxygenase

Nature volume 494, pages 380–384 (2013)Cite this article

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Abstract

Methanotrophs consume methane as their major carbon source and have an essential role in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere1,2,3. These bacteria oxidize methane to methanol by soluble and particulate methane monooxygenases (MMOs)1,2,3,4. Soluble MMO contains three protein components, a 251-kilodalton hydroxylase (MMOH), a 38.6-kilodalton reductase (MMOR), and a 15.9-kilodalton regulatory protein (MMOB), required to couple electron consumption with substrate hydroxylation at the catalytic diiron centre of MMOH2. Until now, the role of MMOB has remained ambiguous owing to a lack of atomic-level information about the MMOH–MMOB (hereafter termed H–B) complex. Here we remedy this deficiency by providing a crystal structure of H–B, which reveals the manner by which MMOB controls the conformation of residues in MMOH crucial for substrate access to the active site. MMOB docks at the α2β2 interface of α2β2γ2 MMOH, and triggers simultaneous conformational changes in the α-subunit that modulate oxygen and methane access as well as proton delivery to the diiron centre. Without such careful control by MMOB of these substrate routes to the diiron active site, the enzyme operates as an NADH oxidase rather than a monooxygenase5. Biological catalysis involving small substrates is often accomplished in nature by large proteins and protein complexes. The structure presented in this work provides an elegant example of this principle.

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Figure 1: MMOB induces conformational changes that affect function.
Figure 2: Conformational changes near the diiron centre and pore residues in MMOH after MMOB binding.
Figure 3: Pore closure and cavity opening after MMOH–MMOB complex formation.
Figure 4: Coordination geometry at the diiron active site of MMOH.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the crystal structure of the H–B complex have been deposited with the Protein Data Bank under the accession code 4GAM.

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Acknowledgements

This work was supported by grant GM 32114 from the National Institute of General Medical Sciences to S. J. Lippard. We thank the staff at the Advanced Light Source beamline 8.2.2. in Lawrence Berkeley National Laboratory for the data collection, S. C. Harrison for resources and comments on the manuscript, and T. C. Johnstone, A. D. Liang and T.-T. Lu for discussions.

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Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Seung Jae Lee, Michael S. McCormick & Stephen J. Lippard

  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Uhn-Soo Cho

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  1. Seung Jae Lee
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  2. Michael S. McCormick
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Contributions

S. J. Lee designed experiments, purified proteins and measured the enzyme activity, analysed data, and wrote the manuscript; M.S.M. analysed the enzyme cavity, analysed data, prepared figures, and wrote the manuscript; S. J. Lippard directed the project, designed experiments, analysed data, and wrote the manuscript; and U.-S.C. obtained crystals, solved and refined the structures, analysed data, and wrote the manuscript. All authors discussed the results and commented on the manuscripts.

Corresponding authors

Correspondence to Stephen J. Lippard or Uhn-Soo Cho.

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The authors declare no competing financial interests.

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Lee, S., McCormick, M., Lippard, S. et al. Control of substrate access to the active site in methane monooxygenase. Nature 494, 380–384 (2013). https://doi.org/10.1038/nature11880

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