Particulate methane monooxygenase (pMMO) is a membranebound 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 Ϸ200 kDa, probably corresponding to an ␣2␤2␥2 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 oxygen͞nitrogen ligands and a 2.57-Å Cu-Cu interaction, providing direct evidence for a copper-containing cluster in pMMO. O ne of the great challenges for the chemical and engineering communities is the selective oxidation of methane to methanol. Although world reserves of petroleum and natural gas are comparable, methane is used far less efficiently as an energy source because it has a low energy density and is hazardous and expensive to transport (1). Conversion of methane to a liquid such as methanol would solve this problem, but current industrial processes for this transformation are costly and inefficient, requiring high temperatures and pressures (2, 3). By contrast, methanotrophic bacteria (4) oxidize methane to methanol at ambient temperature and pressure by using methane monooxygenase (MMO) enzyme systems (5). Only one other enzyme, ammonia monooxygenase (6), can activate the COH bond in methane (104 kcal͞mol). Cytochromes P450 (7) and copper monooxygenases (8) oxidize larger, more reactive hydrocarbons, but cannot hydroxylate methane. Therefore, a detailed understanding of biological methane oxidation is of both fundamental chemical and potential commercial importance.All methanotrophs produce a membrane-bound MMO called particulate MMO (pMMO) (5). Under conditions of low copper availability, several strains also express a soluble enzyme (sMMO) (9), which contains a catalytic diiron center (10). Whereas the structure, biochemistry, and mechanism of sMMO are well understood (11), studies of pMMO are less advanced because of difficulties in solubilizing and purifying active enzyme. The Methylococcus capsulatus (Bath) pMMO comprises three polypeptides, the ␣ (Ϸ47 kDa), ␤ (Ϸ24 kDa), and ␥ (Ϸ22 kDa) subunits, encoded by the pmoB, pmoA, and pmoC genes, respectively (12, 13). It is not known how these three polypeptides are arranged in the pMMO holoenzyme. According to radiolabeling experiments with the suicide substrate acetylene, the active ...

The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-A resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO(iso)), chemically reduced (pMMO(red)), and chemically oxidized (pMMO(ox)) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO(iso) contains both Cu(I) and Cu(II) and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu-Cu interaction in all redox forms of the enzyme. The Cu-Cu distance increases from 2.51 to 2.65 A upon reduction, concomitant with an increase in the average Cu-O/N bond lengths. Appropriate Cu2 model complexes were used to refine and validate the EXAFS fitting protocols for pMMO(iso). Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe(III), is consistent with heme impurities. These findings are complementary to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions.