Raquel L. Lieberman,* Amy C. Rosenzweig,* and Timothy
*Depts. of Biochemistry, Molecular Biology, and Cell Biology
and of Chemistry, Northwestern
University, Evanston, Illinois 60208, USA
#Dept. of Biochemistry and Molecular Biology, Wayne State
University, School of Medicine,
Detroit, Michigan 48201, USA.
Methane-oxidizing bacteria (methanotrophs) are extremely attractive from a
chemist's perspective because these organisms convert methane to methanol. A
detailed understanding of the conversion reaction, which occurs at ambient
temperature and pressure, could lead to new methods for producing methanol, a
potential alternative fuel source. Oxidation of methane is catalyzed by
methane monooxygenase enzymes, which exist in both soluble (sMMO) and
membrane-bound (pMMO) forms.1, 2
While the structure, biochemistry, and
mechanism of sMMO are well understood,3 studies of
pMMO are less advanced due to difficulties in solubilizing and purifying
active protein. The
Methylococcus capsulatus (Bath) pMMO comprises three polypeptides:
a (~47 kDa), b (~24 kDa),
and g (~22 kDa). It is not known how these three
polypeptides are arranged in the pMMO holoenzyme.4,
5 The active site for methane oxidation is
proposed to be located in the b subunit and might
also involve the a subunit.6,
The metal composition of pMMO has been extensively analyzed with varied
results. The enzyme contains primarily copper with metal/protein
stoichiometries ranging from 2 to 15.7-9
Previously published data on
partially purified pMMO indicate bound copper exists as a mixture of the Cu(I)
and Cu(II) oxidation states.10 Based on the
appearance of a type 2 Cu(II) EPR signal, some portion of the copper must
exist in the mononuclear Cu(II) configuration.7, 8 The presence of trinuclear Cu(II)/Cu(I)2 centers in pMMO
was suggested based on XANES analysis and the observation of a hyperfine
splitting pattern of an isotropic EPR signal at g = 2.06.10 In
addition, pMMO often contains a small amount of iron, suggesting the active
site may be heterometallic.7, 8
|Figure 1: XANES Spectra
of the as-isolated (dotted line) and reduced (solid line) pMMO. Dashed line
identifies position of the 1s®4p transition.|
|Figure 2: Fourier Transforms of
as-isolated and reduced pMMO EXAFS Data. Dashed line identified metal-metal
signal in the EXAFS.|
We recently reported the structural characterization of copper centers in
highly purified Methylococcus capsulatus pMMO using X-ray absorption
spectroscopy.11 The as-isolated purified
protein contains a mixture of Cu(I)
and Cu(II), but can easily be reduced using dithionite (Figure 1). Fourier
transforms of EXAFS data for purified and reduced purified pMMO indicate the
presence of a rigid Cu-metal interaction at approximately 2.6 Å in both (Figure
2). Simulations of the EXAFS data for both indicate the presence of a distinct
Cu-Cu interaction in pMMO at 2.57 Å, while the remaining nearest neighbor
ligand environment around the copper is predominately constructed of oxygen and
nitrogen based ligands.
3: Proposed model for the Cu-metal interaction we observe in pMMO. The
identity of the second metal ion has not yet been confirmed and could possibly
be iron. The complex is not necessarily binuclear in metal (i.e., n=1 or 2,
Based on our structural results, we propose the active site of pMMO is
constructed partially (with 50% of the copper) of a multinuclear copper
complex, although the nuclearity of the Cu cluster in pMMO is still unknown.
Based on the short copper-copper distance, the active site in pMMO may resemble
that seen in the catalytic CuZ cluster of nitrous oxide reductase.12 A similar
complex has also been proposed by Lemos et al., in which a symmetric
active site is constructed by two b subunits
Although additional studies
are necessary to elucidate the structural details of the multinuclear center,
these data provide the first evidence for a multinuclear cluster in pMMO.
Murrell, J.C., McDonald, I.R. and Gilbert, B., (2000) Trends
Microbiol., 8, 221-5.
Stanley, S.H., Prior, S.D., Leak, D.J. and Dalton, H., (1983)
Biotechnol. Lett., 6, 487-92.
Merkx, M., D.A., K., Sazinsky, M.H., Blazyk, J.L., Muller, J. and
Lippard, S.J., (2001) Agnew. Chem. Int. Ed., 40, 2782-807.
Stolyar, S., Costello, A.M., Peeples, T.L. and Lidstrom, M.E., (1999)
Microbiology, 145, 1235-44.
Semrau, J.D., Chistoserdov, A., Lebron, J., Costello, A., Davagnino,
J., Kenna, E., Holmes, A.J., Finch, R., Murrell, J.C., and Lidstrom, M.E.,
(1995) J. Bacteriol., 177, 3071-9.
Cook, S.A. and Schiemke, A.K., (1996) J. Inorg. Biochem., 63, 273-84.
Zahn, J.A. and DiSpirito, A.A., (1996) J. Bacteriol., 178, 1018-29.
Basu, P., Katterle, B., Andersson, K.K. and Dalton, H., (2003) Biochem.
J., 369, 412-27.
Nguyen, H.H., Elliott, S.J., Yip, J.H. and Chan, S.I., (1998) J. Biol.
Chem., 273, 7957-66.
Nguyen, H.H.T., Nakagawa, K.H., Hedman, B., Elliott, S.J., Lidstrom,
M.E., Hodgson, K.O., and Chan, S.I., (1996) J. Am. Chem. Soc.,
Lieberman, R.L., Shrestha, D.B., Doan, P.E., Hoffman, B.M., Stemmler,
T.L. and Rosenzweig, A.C., (2003) Proc. Natl. Acad. Sci. USA,
Brown, K., Djinovic-Carugo, K., Haltia, T., Cabrito, I., Saraste, M.,
Moura, J.J., Moura, I., Tegoni, M., and Cambillau, C., (2000) J. Biol.
Chem., 275, 41133-6.
Lemos, S.S., Yuan, H., Collins, M.L.P. and Antholine, W.E., (2002)
Curr. Topics in Biophysics, 26, 43-8.