Chen Chang-Li, Chen Kelvin H-C, Ke Shyue-Chu, Yu Steve S-F, Chan Sunney I
Institute of Chemistry, Academia Sinica, 128 Academia Road Section 2, Nankang, Taipei 115, Taiwan.
J Inorg Biochem. 2004 Dec;98(12):2125-30. doi: 10.1016/j.jinorgbio.2004.09.021.
We report the preparation of a (Cu,Zn)-particulate methane monooxygenase (pMMO) in which the bulk of the copper ions of the electron-transfer clusters (E-clusters) has been replaced by divalent Zn ions. The Cu and Zn contents in the (Cu,Zn)-pMMO were determined by both inductively coupled plasma mass spectroscopy (ICP-MS) and X-ray absorption K-edge spectroscopy. Further characterization of the (Cu,Zn)-pMMO was provided by pMMO-activity assays as well as low-temperature electron paramagnetic resonance (EPR) spectroscopy following reductive titration and incubation in air or air/propylene mixtures. The pMMO-activity assays indicated that the (Cu,Zn)-pMMO was no longer capable of supporting catalytic turnover of hydrocarbon substrates. However, the EPR studies revealed that the catalytic cluster (C-cluster) copper ions in the (Cu,Zn)-pMMO were still capable of supporting the activation of dioxygen when reduced, and that the 14N-superhyperfine features associated with one of the type 2 Cu(II) centers in the hydroxylation C-cluster remained unperturbed. The replacement of the E-cluster copper ions by Zn ions did compromise the ability of the protein to mediate the transfer of reducing equivalents from exogenous reductants to the C-clusters. These observations provide strong support for the electron transfer and catalytic roles for the E-cluster and C-cluster copper ions, respectively.
我们报道了一种（铜，锌）颗粒状甲烷单加氧酶（pMMO）的制备，其中电子转移簇（E簇）中的大部分铜离子已被二价锌离子取代。通过电感耦合等离子体质谱（ICP-MS）和X射线吸收K边光谱法测定了（铜，锌）-pMMO中的铜和锌含量。通过pMMO活性测定以及在空气或空气/丙烯混合物中进行还原滴定和孵育后的低温电子顺磁共振（EPR）光谱对（铜，锌）-pMMO进行了进一步表征。pMMO活性测定表明，（铜，锌）-pMMO不再能够支持烃类底物的催化周转。然而，EPR研究表明，（铜，锌）-pMMO中的催化簇（C簇）铜离子在还原时仍能够支持双氧的活化，并且与羟基化C簇中2型Cu（II）中心之一相关的14N超超精细特征保持不变。用锌离子取代E簇铜离子确实损害了蛋白质将还原当量从外源还原剂转移到C簇的能力。这些观察结果分别为E簇和C簇铜离子的电子转移和催化作用提供了有力支持。
