Lieberman Raquel L, Shrestha Deepak B, Doan Peter E, Hoffman Brian M, Stemmler Timothy L, Rosenzweig Amy C
Department of Biochemistry, Northwestern University, Evanston, IL 60208, USA.
Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3820-5. doi: 10.1073/pnas.0536703100. Epub 2003 Mar 12.
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 approximately 200 kDa, probably corresponding to an alpha(2)beta(2)gamma(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 oxygennitrogen ligands and a 2.57-A Cu-Cu interaction, providing direct evidence for a copper-containing cluster in pMMO.
颗粒性甲烷单加氧酶（pMMO）是一种膜结合酶，可催化嗜甲烷菌中将甲烷氧化为甲醇的反应。了解这种酶如何在常温常压下使甲烷羟化具有重要的基础化学意义和潜在商业价值。然而，由于难以溶解和纯化具有活性的pMMO，导致关于其生化和生物物理性质的报道相互矛盾。我们已从荚膜甲基球菌（巴斯）中纯化出pMMO并检测到了活性。纯化后的酶分子量约为200 kDa，可能对应于α(2)β(2)γ(2)多肽排列。每个200 kDa的pMMO复合物含有4.8±0.8个铜离子和1.5±0.7个铁离子。对应于总铜量40 - 60%的电子顺磁共振光谱参数与单核2型铜位点的存在一致。X射线吸收近边光谱表明纯化后的pMMO是Cu(I)和Cu(II)氧化态的混合物。最后，扩展X射线吸收精细结构数据与氧氮配体以及2.57 Å的Cu - Cu相互作用最匹配，为pMMO中含铜簇提供了直接证据。
