Native top-down mass spectrometry provides insights into the copper centers of membrane-bound methane monooxygenase

Soo Y. Ro, Luis F. Schachner, Christopher W. Koo, Rahul Purohit, Jonathan P. Remis, Grace E. Kenney, Brandon W. Liauw, Paul M. Thomas, Steven M. Patrie, Neil L. Kelleher, Amy C. Rosenzweig

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5 Scopus citations

Abstract

Aerobic methane oxidation is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent, membrane metalloenzyme composed of subunits PmoA, PmoB, and PmoC. Characterization of the copper active site has been limited by challenges in spectroscopic analysis stemming from the presence of multiple copper binding sites, effects of detergent solubilization on activity and crystal structures, and the lack of a heterologous expression system. Here we utilize nanodiscs coupled with native top-down mass spectrometry (nTDMS) to determine the copper stoichiometry in each pMMO subunit and to detect post-translational modifications (PTMs). These results indicate the presence of a mononuclear copper center in both PmoB and PmoC. pMMO-nanodisc complexes with a higher stoichiometry of copper-bound PmoC exhibit increased activity, suggesting that the PmoC copper site plays a role in methane oxidation activity. These results provide key insights into the pMMO copper centers and demonstrate the ability of nTDMS to characterize complex membrane-bound metalloenzymes.

Original languageEnglish (US)
Article number2675
JournalNature communications
Volume10
Issue number1
DOIs
StatePublished - Dec 1 2019

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ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physics and Astronomy(all)

Cite this

Ro, S. Y., Schachner, L. F., Koo, C. W., Purohit, R., Remis, J. P., Kenney, G. E., Liauw, B. W., Thomas, P. M., Patrie, S. M., Kelleher, N. L., & Rosenzweig, A. C. (2019). Native top-down mass spectrometry provides insights into the copper centers of membrane-bound methane monooxygenase. Nature communications, 10(1), [2675]. https://doi.org/10.1038/s41467-019-10590-6