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

Authors :
Pham MD
Lin YP
Van Vuong Q
Nagababu P
Chang BT
Ng KY
Chen CH
Han CC
Chen CH
Li MS
Yu SS
Chan SI
Source :
Biochimica et biophysica acta [Biochim Biophys Acta] 2015 Dec; Vol. 1854 (12), pp. 1842-1852. Date of Electronic Publication: 2015 Aug 11.
Publication Year :
2015

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 (C2H2O) 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 are controlled tightly within the transmembrane domain. Support of these conclusions is provided by parallel experiments with two related alkynes: propyne (CH3CCH) and trifluoropropyne (CF3CCH). Finally, we discuss the implication of these findings to the location of the catalytic site in pMMO.<br /> (Copyright © 2015 Elsevier B.V. All rights reserved.)

Details

Language :
English
ISSN :
0006-3002
Volume :
1854
Issue :
12
Database :
MEDLINE
Journal :
Biochimica et biophysica acta
Publication Type :
Academic Journal
Accession number :
26275807
Full Text :
https://doi.org/10.1016/j.bbapap.2015.08.004