The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein

Basu, Piku; Katterle, Bettina; Andersson, K. Kristoffer; Dalton, Howard

doi:10.1042/bj20020823

Cited by 97 publications

(191 citation statements)

References 45 publications

(92 reference statements)

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“…The addition of 0.2-72 mM EDTA decreased activity, but approximately half of the original specific activity remained even with a 40-fold molar excess of EDTA. A similar effect has been observed for purified pMMO from M. trichosporium OB3b (18) and for partially purified pMMO from M. capsulatus (Bath) (16). These observations suggest that the metal ions in pMMO are necessary for activity, but may differ in accessibility.…”

Section: Molecular Mass Determinationsupporting

confidence: 66%

“…The average specific activity of the membranebound pMMO is 19.4 nmol͞mg⅐min, comparable to other preparations (14,16,17,19,20) (Table 1). For membrane-bound pMMO, both NADH and duroquinol (32) were effective reducing agents.…”

Section: Molecular Mass Determinationsupporting

confidence: 48%

“…Our purification protocol differs from those reported (14,16,17). The Source 30Q anion exchange column removes contaminating proteins as well as lipids.…”

Section: Resultsmentioning

confidence: 53%

“…By contrast, other researchers observe only a typical type 2 Cu(II) EPR signal for purified M. capsulatus (Bath) pMMO (14,16) and for membrane-bound and whole-cell suspensions of M. capsulatus (Bath) and Methylomicrobium album BG8 pMMO (22)(23)(24). Antholine and coworkers (22) suggest that the isotropic signal attributed to a trinuclear cluster can instead be explained by the presence of a radical and a mononuclear Cu(II) ion coordinated by three or four nitrogen donors in a square planar geometry.…”

mentioning

confidence: 99%

“…Antholine and coworkers (22) suggest that the isotropic signal attributed to a trinuclear cluster can instead be explained by the presence of a radical and a mononuclear Cu(II) ion coordinated by three or four nitrogen donors in a square planar geometry. In addition to copper, 1-2.5 iron ions have been detected in some preparations of purified pMMO from both M. capsulatus (Bath) (14,16) and M. trichosporium OB3b (18). To address some of the many unresolved issues regarding pMMO, we have purified the enzyme from M. capsulatus (Bath), determined its molecular mass and metal ion content, and investigated the metal centers by x-ray absorption (XAS) and EPR spectroscopies.…”

mentioning

confidence: 99%

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Purified particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a copper-containing cluster

Lieberman

Shrestha

Doan

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

146

207

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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 ...

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Section: Molecular Mass Determinationsupporting

confidence: 66%

Section: Molecular Mass Determinationsupporting

confidence: 48%

“…Our purification protocol differs from those reported (14,16,17). The Source 30Q anion exchange column removes contaminating proteins as well as lipids.…”

Section: Resultsmentioning

confidence: 53%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Purified particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a copper-containing cluster

Lieberman

Shrestha

Doan

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

146

207

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Particulate Methane Monooxygenase

Rosenzweig

2004

Handbook of Metalloproteins

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Particulate methane monooxygenase (pMMO) catalyzes the oxidation of methane to methanol in methanotrophic bacteria. The 300‐kDa pMMO enzyme is a trimeric integral membrane protein, comprising three copies each of three subunits: pmoB, pmoA, and pmoC. Purified pMMO contains both copper and iron, although some of the iron may be due to heme contamination. The copper stoichiometry ranges from 2 to 15 copper ions per 100‐kDa pMMO protomer, depending on the enzyme source. EPR spectra of whole cells, membrane‐bound, and purified pMMO consistently indicate the presence of type 2 Cu(II). According to XAS data, both Cu(I) and Cu(II) are present. EXAFS data reveal the presence of oxygen/nitrogen ligands and a short CuCu interaction at 2.5 Å that increases to 2.6 Å upon chemical reduction. Crystal structures of pMMO from two organisms have been reported. A soluble region consisting of six cupredoxin‐like β‐barrel structures is supported by 15 transmembrane helices. Three different metal centers have been detected crystallographically: a highly conserved dinuclear copper center, a nonconserved mononuclear copper center, and a site that can be occupied by zinc or copper. Although the identity of the pMMO active site remains unknown, some information regarding the mechanism has been obtained from substrate studies and computational work.

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Copper Enzymes/Models

Park¹,

Lee²,

Karlin³

2010

Encyclopedia of Catalysis

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Copper is utilized as a catalytic metal center in a variety of enzymes involved in oxidation–reduction processes. The Cu(I) and Cu(II) oxidation states are readily accessible and interconvertible under physiological conditions via oxidants (ie, O 2 ) or reducing agents such as ascorbic acid or glutathione. The important chemical transformations that these enzymes are responsible for include dioxygen processing as well as nitrogen oxide (NOx) conversions. The active site structures and catalytic mechanisms of the most well‐studied copper enzymes are described. The contributions to the understanding of enzyme function obtained from biomimetic studies using synthetic copper complexes of most relevance to the active site features have also been highlighted.

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The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein

Cited by 97 publications

References 45 publications

Purified particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a copper-containing cluster

Purified particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a copper-containing cluster

Particulate Methane Monooxygenase

Copper Enzymes/Models

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