Chan Sunney I, Chen Kelvin H-C, Yu Steve S-F, Chen Chang-Li, Kuo Simon S-J
Institute of Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan.
Biochemistry. 2004 Apr 20;43(15):4421-30. doi: 10.1021/bi0497603.
The particulate methane monooxygenase (pMMO) is a complex membrane protein complex that has been difficult to isolate and purify for biochemical and biophysical characterization because of its instability in detergents used to solubilize the enzyme. In this perspective, we summarize the progress recently made toward obtaining a purified pMMO-detergent complex and characterizing the enzyme in pMMO-enriched membranes. The purified pMMO is a multi-copper protein, with ca. 15 copper ions sequestered into five trinuclear copper clusters: two for dioxygen chemistry and alkane hydroxylation (catalytic or C-clusters) and three to provide a buffer of reducing equivalents to re-reduce the C-clusters following turnover (electron transfer or E-clusters). The enzyme is functional when all the copper ions are reduced. When the protein is purified under ambient aerobic conditions in the absence of a hydrocarbon substrate, only the C-clusters are oxidized; there is an apparent kinetic barrier for electron transfer from the E-cluster copper ions to the C-clusters under these conditions. Evidence is provided in support of both C-clusters participating in the dioxygen chemistry, but only one C-cluster supporting alkane hydroxylation. Acetylene modification of the latter C-cluster in the hydrophobic pocket of the active site lowers or removes the kinetic barrier for electron transfer from the E-clusters to the C-clusters so that all the copper ions could be fully oxidized by dioxygen. A model for the hydroxylation chemistry when a hydrocarbon substrate is bound to the active site of the hydroxylation C-cluster is presented. Unlike soluble methane monooxygenase (sMMO), pMMO exhibits limited substrate specificity, but the hydroxylation chemistry is highly regioselective and stereoselective. In addition, the hydroxylation occurs with total retention of configuration of the carbon center that is oxidized. These results are consistent with a concerted mechanism involving direct side-on insertion of an active singlet "oxene" from the activated copper cluster across the "C-H" bond in the active site. Finally, in our hands, both the purified pMMO-detergent complex and pMMO-enriched membranes exhibit high NADH-sensitive as well as duroquinol-sensitive specific activity. A possible role for the two reductants in the turnover of the enzyme is proposed.
颗粒性甲烷单加氧酶（pMMO）是一种复杂的膜蛋白复合物，由于其在用于溶解该酶的去污剂中不稳定，一直难以分离和纯化以进行生化和生物物理特性分析。从这个角度出发，我们总结了最近在获得纯化的pMMO - 去污剂复合物以及表征富含pMMO的膜中的该酶方面所取得的进展。纯化后的pMMO是一种多铜蛋白，约有15个铜离子螯合形成五个三核铜簇：两个用于双加氧反应和烷烃羟基化（催化或C簇），三个用于提供还原当量缓冲，以便在周转后重新还原C簇（电子转移或E簇）。当所有铜离子都被还原时，该酶具有活性。当在没有烃类底物的环境需氧条件下纯化该蛋白时，只有C簇被氧化；在这些条件下，从E簇铜离子到C簇的电子转移存在明显的动力学障碍。有证据支持两个C簇都参与双加氧反应，但只有一个C簇支持烷烃羟基化。活性位点疏水口袋中后一个C簇的乙炔修饰降低或消除了从E簇到C簇的电子转移动力学障碍，从而使所有铜离子都能被双加氧完全氧化。本文提出了一种烃类底物与羟基化C簇的活性位点结合时的羟基化反应化学模型。与可溶性甲烷单加氧酶（sMMO）不同，pMMO表现出有限的底物特异性，但羟基化反应具有高度的区域选择性和立体选择性。此外，羟基化反应发生时，被氧化的碳中心的构型完全保留。这些结果与一种协同机制一致，该机制涉及活性单重态“氧烯”从活化的铜簇直接侧向插入活性位点的“C - H”键。最后，在我们的研究中，纯化的pMMO - 去污剂复合物和富含pMMO的膜都表现出对NADH敏感以及对硬脂酰辅酶Q敏感的比活性。我们提出了这两种还原剂在该酶周转中的可能作用。
