Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene.
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.
Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan; Taiwan International Graduate Program (TIGP), Academia Sinica, Taipei 11529, Taiwan; Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan.
Minh D
MD
Pham
Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan.
Ya-Ping
YP
Lin
Institute for Computational Science and Technology, Ho Chi Minh City, Vietnam.
Quan
Q
Van Vuong
Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Penumaka
P
Nagababu
Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Brian T-A
BT
Chang
Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Kok Yaoh
KY
Ng
Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan.
Chein-Hung
CH
Chen
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan.
Chau-Chung
CC
Han
Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan.
Chung-Hsuan
CH
Chen
Institute for Computational Science and Technology, Ho Chi Minh City, Vietnam; Institute of Physics, Polish Academy of Sciences, 02-668 Warsaw, Poland.
Mai Suan
MS
Li
Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan. Electronic address: sfyu@gate.sinica.edu.tw.
Steve S-F
SS
Yu
Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan; Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan; Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA. Electronic address: sunneychan@yahoo.com.
Sunney I
SI
Chan
D000114:Acetylene / Q000378:metabolism*; D002853:Chromatography, Liquid; D020619:Methylococcus capsulatus / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D010105:Oxygenases; C021555:methane monooxygenase; D000114:Acetylene
Acetylene; Computational simulation; Mass spectrometry; Mechanism-based inactivation; Methylococcus capsulatus (Bath); Particulate methane monooxygenase