Purified Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) Is a Dimer with Both Mononuclear Copper and a Copper-Containing Cluster

Particulate methane monooxygenase (pMMO) is a membrane-bound enzyme that catalyzes the oxidation of methane to methanol in methanotropic bacteria. Understanding how this enzyme hydroxylates methane at ambient temperature and pressure is of fundamental chemical and potential commercial importance. Di...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2003-04, Vol.100 (7), p.3820-3825
Main Authors: Lieberman, Raquel L., Shrestha, Deepak B., Doan, Peter E., Hoffman, Brian M., Stemmler, Timothy L., Rosenzweig, Amy C.
Format: Article
Language:English
Subjects:
Biochemistry
Bioinorganic Chemistry Special Feature
Biological Sciences
Cell Fractionation - methods
Cell Membrane - enzymology
Circular Dichroism
Copper
Copper - analysis
Dimerization
Electron Spin Resonance Spectroscopy
Enzymes
Gels
Ions
Kinetics
Metal ions
Metalloproteins - chemistry
Metalloproteins - isolation & purification
Metalloproteins - metabolism
Methane
Methylococcus capsulatus - enzymology
Molecular Weight
Oxidation
Oxygen
Oxygenases - chemistry
Oxygenases - isolation & purification
Oxygenases - metabolism
Propylene
Protein Conformation
Spectroscopy
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Summary:Particulate methane monooxygenase (pMMO) is a membrane-bound enzyme that catalyzes the oxidation of methane to methanol in methanotropic bacteria. Understanding how this enzyme hydroxylates methane at ambient temperature and pressure is of fundamental chemical and potential commercial importance. Difficulties in solubilizing and purifying active pMMO have led to conflicting reports regarding its biochemical and biophysical properties, however. We have purified pMMO from Methylococcus capsulatus (Bath) and detected activity. The purified enzyme has a molecular mass of ≈200 kDa, probably corresponding to an α2 β2 γ2 polypeptide arrangement. Each 200-kDa pMMO complex contains 4.8 ± 0.8 copper ions and 1.5 ± 0.7 iron ions. Electron paramagnetic resonance spectroscopic parameters corresponding to 40-60% of the total copper are consistent with the presence of a mononuclear type 2 copper site. X-ray absorption near edge spectra indicate that purified pMMO is a mixture of Cu(I) and Cu(II) oxidation states. Finally, extended x-ray absorption fine structure data are best fit with oxygen/nitrogen ligands and a 2.57-A Cu-Cu interaction, providing direct evidence for a copper-containing cluster in pMMO.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0536703100