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Originally published In Press as doi:10.1074/jbc.M107712200 on November 14, 2001

J. Biol. Chem., Vol. 277, Issue 8, 5858-5865, February 22, 2002
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Why OrfY?
CHARACTERIZATION OF MMOD, A LONG OVERLOOKED COMPONENT OF THE SOLUBLE METHANE MONOOXYGENASE FROM METHYLOCOCCUS CAPSULATUS (BATH)*,

Maarten MerkxDagger and Stephen J. Lippard§

From the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Soluble methane monooxygenase (sMMO) has been studied intensively to understand the mechanism by which it catalyzes the remarkable oxidation of methane to methanol. The cluster of genes that encode for the three characterized protein components of sMMO (MMOH, MMOB, and MMOR) contains an additional open reading frame (orfY) of unknown function. In the present study, MMOD, the protein encoded by orfY, was overexpressed as a fusion protein in Escherichia coli. Pure MMOD was obtained in high yields after proteolytic cleavage and a two-step purification procedure. Western blot analysis of Methylococcus capsulatus (Bath) soluble cell extracts showed that MMOD is expressed in the native organism although at significantly lower levels than the other sMMO proteins. The cofactorless MMOD protein is a potent inhibitor of sMMO activity and binds to the hydroxylase protein (MMOH) with an affinity similar to that of MMOB and MMOR. The addition of up to 2 MMOD per MMOH results in changes in the optical spectrum of the hydroxylase that suggest the formation of a (µ-oxo)diiron(III) center in a fraction of the MMOH-MMOD complexes. Possible functions for MMOD are discussed, including a role in the assembly of the MMOH diiron center similar to that suggested for DmpK, a protein that shares some properties with MMOD.

* This work was supported by a grant from the National Institute of General Medical Sciences.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.

The on-line version of this article (available at http://www.jbc.org) contains Tables S1 and S2, Figs. S1 and S2 and Schemes S1 and S2.

Dagger A Human Frontier of Science Program postdoctoral fellow.

§ To whom correspondence should be addressed: Dept. of Chemistry, Rm. 18-T122, Massachusetts Institute of Technology, Cambridge, MA 02139. Fax: 617-258-8150; E-mail: lippard@lippard.mit.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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