Low-Frequency EPR of the Copper in Particulate Methane Monooxygenase from <i>Methylomicrobium </i><i>albus</i> BG8

Yuan, Hua; Collins, Mary Lynne Perille; Antholine, William E.

doi:10.1021/ja9701669

Cited by 47 publications

(64 citation statements)

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“…3). These parameters are similar to those reported for both whole-cell suspensions (24) and purified samples (14, 16) from M. capsulatus (Bath) and are consistent with a mononuclear Cu(II) site coordinated by three or four histidine imidazoles (22)(23)(24)39). By contrast, the signal attributed by Chan and coworkers (17,19,20) to a mixed valence trinuclear copper cluster is not observed in either spectrum.…”

Section: Molecular Mass Determinationsupporting

confidence: 87%

“…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).…”

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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: 87%

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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“…The results also suggest that Cu-mb is the probable source of the variability, complexity and controversy associated with the copper centres of the pMMO (Basu et al, 2003;Chan et al, 2004;Choi et al, 2003;Lieberman et al, 2003;Lieberman & Rosenzweig, 2004Nguyen et al, 1994Nguyen et al, , 1996Nguyen et al, , 1998Takeguchi & Okura, 2000;Takeguchi et al, 1999;Téllez et al, 1998;Yuan et al, 1997Yuan et al, , 1998aYuan et al, , b, 1999Zahn & DiSpirito, 1996). Laboratories examining the pMMO have differed in the analysis of the EPR spectra.…”

Section: Discussionmentioning

confidence: 89%

“…One surprising result in the examination of membrane samples was the absence of the normally well-resolved superhyperfine structure in the g H region associated with the type II Cu(II) of the pMMO (Nguyen et al, 1996;Yuan et al, 1997, 1998a, b, 1999]. This phenomenon was only observed in membrane preparations from cells cultured in high-copper, i.e.…”

Section: Effects Of Mb On the Cupric Signal In The G H Regionmentioning

confidence: 99%

Effect of methanobactin on the activity and electron paramagnetic resonance spectra of the membrane-associated methane monooxygenase in Methylococcus capsulatus Bath

et al. 2005

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Improvements in the purification of methanobactin (mb) from either Methylosinus trichosporium OB3bT or Methylococcus capsulatus Bath resulted in preparations that stimulated methane-oxidation activity in both whole-cell and cell-free fractions of Methylococcus capsulatus Bath expressing the membrane-associated methane monooxygenase (pMMO). By using washed membrane factions with pMMO activities in the 290 nmol propylene oxidized min "1 (mg protein) "1 range, activities approaching 400 nmol propylene oxidized min "1 (mg protein) "1 were commonly observed following addition of copper-containing mb (Cu-mb), which represented 50-75 % of the total whole-cell activity.The stimulation of methane-oxidation activity by Cu-mb was similar to or greater than that observed with equimolar concentrations of Cu(II), without the inhibitory effects observed with high copper concentrations. Stimulation of pMMO activity was not observed with copper-free mb, nor was it observed when the copper-to-mb ratio was <0?5 Cu atoms per mb. The electron paramagnetic resonance (EPR) spectra of mb differed depending on the copper-to-mb ratio. At copper-to-mb ratios of <0?4 Cu(II) per mb, Cu(II) addition to mb showed an initial coordination by both sulfur and nitrogen, followed by reduction to Cu(I) in <2 min. At Cu(II)-to-mb ratios between 0?4 and 0?9 Cu(II) per mb, the intensity of the Cu(II) signal in EPR spectra was more representative of the Cu(II) added and indicated more nitrogen coordination. The EPR spectral properties of mb and pMMO were also examined in the washed membrane fraction following the addition of Cu(II), mb and Cu-mb in the presence or absence of reductants (NADH or duroquinol) and substrates (CH 4 and/or O 2 ). The results indicated that Cu-mb increased electron flow to the pMMO, increased the free radical formed following the addition of O 2 and decreased the residual free radical following the addition of O 2 plus CH 4 . The increase in pMMO activity and EPR spectral changes to the pMMO following Cu-mb addition represent the first positive evidence of interactions between the pMMO and Cu-mb.

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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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Low-Frequency EPR of the Copper in Particulate Methane Monooxygenase from Methylomicrobium albus BG8

Cited by 47 publications

References 15 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

Effect of methanobactin on the activity and electron paramagnetic resonance spectra of the membrane-associated methane monooxygenase in Methylococcus capsulatus Bath

Particulate Methane Monooxygenase

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