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Toward Delineating the Structure and Function of the Particulate Methane Monooxygenase from Methanotrophic Bacteria

Chan, Sunney I. and Chen, Kelvin H.-C. and Yu, Steve S.-F. and Chen, Chang-Li and Kuo, Simon S.-J. (2004) Toward Delineating the Structure and Function of the Particulate Methane Monooxygenase from Methanotrophic Bacteria. Biochemistry, 43 (15). pp. 4421-4430. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20170131-140941315

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Abstract

The particulate methane monooxygenase (pMMO) is a complex membrane protein complex that has been difficult to isolate and purify for biochemical and biophysical characterization because of its instability in detergents used to solubilize the enzyme. In this perspective, we summarize the progress recently made toward obtaining a purified pMMO−detergent complex and characterizing the enzyme in pMMO-enriched membranes. The purified pMMO is a multi-copper protein, with ca. 15 copper ions sequestered into five trinuclear copper clusters:  two for dioxygen chemistry and alkane hydroxylation (catalytic or C-clusters) and three to provide a buffer of reducing equivalents to re-reduce the C-clusters following turnover (electron transfer or E-clusters). The enzyme is functional when all the copper ions are reduced. When the protein is purified under ambient aerobic conditions in the absence of a hydrocarbon substrate, only the C-clusters are oxidized; there is an apparent kinetic barrier for electron transfer from the E-cluster copper ions to the C-clusters under these conditions. Evidence is provided in support of both C-clusters participating in the dioxygen chemistry, but only one C-cluster supporting alkane hydroxylation. Acetylene modification of the latter C-cluster in the hydrophobic pocket of the active site lowers or removes the kinetic barrier for electron transfer from the E-clusters to the C-clusters so that all the copper ions could be fully oxidized by dioxygen. A model for the hydroxylation chemistry when a hydrocarbon substrate is bound to the active site of the hydroxylation C-cluster is presented. Unlike soluble methane monooxygenase (sMMO), pMMO exhibits limited substrate specificity, but the hydroxylation chemistry is highly regioselective and stereoselective. In addition, the hydroxylation occurs with total retention of configuration of the carbon center that is oxidized. These results are consistent with a concerted mechanism involving direct side-on insertion of an active singlet “oxene” from the activated copper cluster across the “C−H” bond in the active site. Finally, in our hands, both the purified pMMO−detergent complex and pMMO-enriched membranes exhibit high NADH-sensitive as well as duroquinol-sensitive specific activity. A possible role for the two reductants in the turnover of the enzyme is proposed.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bi0497603DOIArticle
http://pubs.acs.org/doi/full/10.1021/bi0497603PublisherArticle
ORCID:
AuthorORCID
Chan, Sunney I.0000-0002-5348-2723
Yu, Steve S.-F.0000-0002-3462-065X
Additional Information:© 2004 American Chemical Society. Received 3 February 2004. Published online 19 March 2004. Published in print 1 April 2004. This work was supported by grants from the National Science Council (NSC 91-2113-M-001-045 and 92-2113-M-001-057) and the National Institute of General Medical Sciences, U.S. Public Health Service (GM22432).
Funders:
Funding AgencyGrant Number
National Science Council (Taipei)NSC 91-2113-M-001-045
National Science Council (Taipei)92-2113-M-001-057
NIHGM22432
Issue or Number:15
Record Number:CaltechAUTHORS:20170131-140941315
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170131-140941315
Official Citation:Toward Delineating the Structure and Function of the Particulate Methane Monooxygenase from Methanotrophic Bacteria Sunney I. Chan, Kelvin H.-C. Chen, Steve S.-F. Yu, Chang-Li Chen, and Simon S.-J. Kuo Biochemistry 2004 43 (15), 4421-4430 DOI: 10.1021/bi0497603
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73891
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:01 Feb 2017 20:36
Last Modified:09 Mar 2020 13:19

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