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Product analogue binding identifies the copper active site of particulate methane monooxygenase

Nature Catalysis volume 6, pages 1194–1204 (2023)Cite this article

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Abstract

Nature’s primary methane-oxidizing enzyme, the membrane-bound particulate methane monooxygenase (pMMO), catalyses the oxidation of methane to methanol. Copper is required for pMMO activity, and decades of structural and spectroscopic studies have sought to identify the active site among three candidates: the CuB, CuC and CuD sites. Challenges associated with the isolation of active pMMO hindered identification of its catalytic centre; however, we have recently shown that reconstituting pMMO into native lipid nanodiscs stabilizes its structure and restores its activity. Here, such active samples were incubated with 2,2,2-trifluoroethanol, a product analogue that serves as a readily visualized active-site probe. Interactions between 2,2,2-trifluoroethanol and the CuD site were observed with pulsed electron nuclear double resonance spectroscopy and cryoelectron microscopy, implicating CuD and the surrounding hydrophobic pocket as the likely site of methane oxidation. Use of these orthogonal techniques on parallel samples is a powerful approach that can circumvent difficulties in interpreting metalloenzyme cryoelectron microscopy maps.

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Fig. 1: CryoEM structure of M. capsulatus (Bath) pMMO in native lipid nanodiscs.
Fig. 2: Parallel EPR and cryoEM studies of pMMO in native lipid nanodiscs.
Fig. 3: ENDOR spectroscopic analysis of TFE interacting with the pMMO copper sites.
Fig. 4: ENDOR detection of a specific interaction between the pMMO CuD site and TFE.
Fig. 5: CryoEM structures of pMMO in native lipid nanodiscs with 0× and 20× TFE.

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Data availability

The models of pMMO in native lipid nanodiscs (7S4H), for KCN-treated (8SR5), KCN-treated and copper-reloaded (8SR4), 20× TFE (8OYI), cross-linked 20× TFE (8SQW), 20× TFB (8SR2) and cross-linked 20× TFB (8SR1) samples are available in the Protein Data Bank (PDB). Corresponding cryoEM maps are available at the Electron Microscopy Data Bank (EMDB). Other data are available in the main text, Supplementary Information or from the authors on reasonable request. Source Data are provided with this paper.

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Acknowledgements

This work was supported by NIH grants R35GM118035 (A.C.R.), R01GM111097 (B.M.H.), T32GM105538 (F.J.T.), F31ES034283 (F.J.T.), and T32GM008382 (R.J.J.), as well as the NSF MCB-1908587 (B.M.H) This work used resources of the Northwestern Structural Biology Facility and the Northwestern Keck Biophysics Facility, which are supported by the NCI CCSG P30 CA060553 grant awarded to the Robert H. Lurie Comprehensive Cancer Center. Metal analysis was performed at the Northwestern University Quantitative Bio-element Imaging Center generously supported by NASA Ames Research Center NNA06CB93G. Some of this work was performed at the National Center for CryoEM Access and Training (NCCAT) and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24 GM129539) and by grants from the Simons Foundation (SF349247) and NY State Assembly. We thank L. Mazhar for experimental assistance, Y. He for guidance in cryoEM, P. Doan for helpful discussions, J. Pattie for computer-related support and M. Ho for assistance during revisions.

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  1. These authors contributed equally: Frank J. Tucci, Richard J. Jodts.

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  1. Departments of Molecular Biosciences and of Chemistry, Northwestern University, Evanston, IL, USA

    Frank J. Tucci, Richard J. Jodts, Brian M. Hoffman & Amy C. Rosenzweig

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Contributions

F.J.T., R.J.J., B.M.H. and A.C.R. conceptualized the work and designed experiments. F.J.T. and R.J.J. performed experiments, analysed data and prepared figures. F.J.T., R.J.J., B.M.H. and A.C.R. contributed to writing and editing the manuscript.

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Correspondence to Brian M. Hoffman or Amy C. Rosenzweig.

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Tucci, F.J., Jodts, R.J., Hoffman, B.M. et al. Product analogue binding identifies the copper active site of particulate methane monooxygenase. Nat Catal 6, 1194–1204 (2023). https://doi.org/10.1038/s41929-023-01051-x

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