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The Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) Is a Novel Copper-containing Three-subunit Enzyme: isolation and charactization

Nguyen, Hiep-Hoa T. and Elliott, Sean J. and Yip, John Hon-Kay and Chan, Sunney I. (1998) The Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) Is a Novel Copper-containing Three-subunit Enzyme: isolation and charactization. Journal of Biological Chemistry, 273 (14). pp. 7957-7966. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:NGUjbc98

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Abstract

The particulate methane monooxygenase (pMMO) is known to be very difficult to study mainly due to its unusual activity instability in vitro. By cultivating Methylococcus capsulatus (Bath) under methane stress conditions and high copper levels in the growth medium, membranes highly enriched in the pMMO with exceptionally stable activity can be isolated from these cells. Purified and active pMMO can be subsequently obtained from these membrane preparations using protocols in which an excess of reductants and anaerobic conditions were maintained during membrane solubilization by dodecyl beta-D-maltoside and purification by chromatography. The pMMO was found to be the major constituent in these membranes, constituting 60-80% of total membrane proteins. The dominant species of the pMMO was found to consist of three subunits, alpha, beta, and gamma, with an apparent molecular mass of 45, 26, and 23 kDa, respectively. A second species of the pMMO, a proteolytically processed version of the enzyme, was found to be composed of three subunits, alpha', beta, and gamma, with an apparent molecular mass of 35, 26, and 23 kDa, respectively. The alpha and alpha' subunits from these two forms of the pMMO contain identical N-terminal sequences. The gamma subunit, however, exhibits variation in its N-terminal sequence. The pMMO is a copper-containing protein only and shows a requirement for Cu(I) ions. Approximately 12-15 Cu ions per 94-kDa monomeric unit were observed. The pMMO is sensitive to dioxygen tension. On the basis of dioxygen sensitivity, three kinetically distinct forms of the enzyme can be distinguished. A slow but air-stable form, which is converted into a "pulsed" state upon direct exposure to atmospheric oxygen pressure, is considered as type I pMMO. This form was the subject of our pMMO isolation effort. Other forms (types II and III) are deactivated to various extents upon exposure to atmospheric dioxygen pressure. Under inactivating conditions, these unstable forms release protons to the buffer (~10 H+/94-kDa monomeric unit) and eventually become completely inactive.


Item Type:Article
Additional Information:Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. (Received for publication, July 7, 1997, and in revised form, December 22, 1997) We thank Prof. Mary E. Lidstrom and Drs. Andrei Chistoserdov, Ludmila Chistoserdova, and Roopa Ramamorthi for helpful discussions and Dr. Peter Green for assistance with the inductively coupled plasma-mass spectroscopy analysis. This work was supported by NIGMS, National Institutes of Health, Grant GM 22432 (to S.I.C.). Unrestricted financial support was also received from the George Grant Hoag Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. [H.-H.T.N. was the] [r]ecipient of a W. R. Grace fellowship and a National Research Service Predoctoral Award.
Record Number:CaltechAUTHORS:NGUjbc98
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:NGUjbc98
Alternative URL:http://www.jbc.org/cgi/content/abstract/273/14/7957
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10948
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:20 Jun 2008
Last Modified:26 Dec 2012 10:06

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