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Published December 2015 | Supplemental Material
Journal Article Open

Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene

Abstract

Acetylene (HCCH) has a long history as a mechanism-based enzyme inhibitor and is considered an active-site probe of the particulate methane monooxygenase (pMMO). Here, we report how HCCH inactivates pMMO in Methylococcus capsulatus (Bath) by using high-resolution mass spectrometry and computational simulation. High-resolution MALDI-TOF MS of intact pMMO complexes has allowed us to confirm that the enzyme oxidizes HCCH to the ketene (C_2H_2O) intermediate, which then forms an acetylation adduct with the transmembrane PmoC subunit. LC-MS/MS analysis of the peptides derived from in-gel proteolytic digestion of the protein subunit identifies K196 of PmoC as the site of acetylation. No evidence is obtained for chemical modification of the PmoA or PmoB subunit. The inactivation of pMMO by a single adduct in the transmembrane PmoC domain is intriguing given the complexity of the structural fold of this large membrane-protein complex as well as the complicated roles played by the various metal cofactors in the enzyme catalysis. Computational studies suggest that the entry of hydrophobic substrates to, and migration of products from, the catalytic site of pMMO is controlled tightly within the transmembrane domain. Support of these conclusions is provided by parallel experiments with two related alkynes: propyne (CH3CCH) and trifluoropropyne (CF_3CCH). Finally, we discuss the implication of these findings to the location of the catalytic site in pMMO.

Additional Information

© 2015 Elsevier B.V. Received date: 16 June 2015; Revised date: 29 July 2015; Accepted date: 9 August 2015. This work was supported by Academia Sinica and by research grants from the National Science Council of the Republic of China (NSC 98-2113-M-001-026; NSC 99-2119-M-001-003; NSC 100-2113-M-001-002; and 101-2113-M-001-013- to SIC; and NSC 101-2113-M-001-007-MY3 to SSFY). The computer simulations were supported by the Department of Science and Technology at Ho Chi Minh City, Vietnam and Narodowe Centrum Nauki in Poland (grant 2011/01/B/NZ1/01622 to MSL). We acknowledge numerous scientific discussions on this work with Dr. Chia-Min Yang (Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan). M.D.P. is also grateful to Dr. Yang for serving as his academic advisor.

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August 22, 2023
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