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研究生:陳昌立
研究生(外文):Chang-Li Chen
論文名稱:銅離子與嗜甲烷菌Methylococcuscapsulatus(Bath)中之微粒體甲烷單氧化酵素
論文名稱(外文):Copper Ions and The Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath)
指導教授:陳長謙陳長謙引用關係
指導教授(外文):Sunney I. Chan
學位類別:博士
校院名稱:國立清華大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:103
中文關鍵詞:嗜甲烷菌銅離子電子自旋光譜
外文關鍵詞:pMMOcopperEPR
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銅離子在嗜甲烷菌 Methylococcus capsulatus (Bath) 的微粒體甲烷單氧化酵素中扮演著不可獲缺的重要角色。一個甲烷單氧化酵素中含有十五個呈還原態的銅離子,而且這些銅離子排列成五個三核銅離子簇(trinuclear copper cluster)。其中兩個三核銅離子簇稱為“基質催化銅離子簇”(C-cluster),它們參與了氧氣的鍵結與轉化以及碳氫化合物受質的羥化反應。剩下的銅離子簇稱為“電子傳遞銅離子簇”(E-cluster),它們負責將電子由NADH傳送給“基質催化銅離子簇”。初步分離的微粒狀甲烷單氧化酵素其低溫電子自旋光譜含有兩組訊號:一組屬於“第二型銅離子”(type 2 copper) ,另一組則是屬於三核銅離子簇。經過計算模擬,我們發現這組三核銅離子簇的電子自旋光譜訊號其電子自旋偶合常數J 約為 15�{20 cm-1,零場分裂常數D 及E約為0.0175 cm-1及0.005 cm-1。藉著結合低溫電子自旋光譜以及快速低溫擷取的技術,我們成功地觀察到甲烷單氧化酵素在催化循環過程中的不同氧化態,並證實了此酵素的催化反應機制。
利用傳統發酵系統與微管束型過濾器的結合,我們成功的培養出銅鋅混合型甲烷單氧化酵素。藉著感應偶合電漿質譜儀與X射線吸收光譜儀的測定,我們發現每一個銅鋅混合型甲烷單氧化酵素約含有七個銅與六個鋅。除此之外,我們亦利用低溫電子自旋光譜進一步鑑定此酵素的結構與性質。我們發現銅鋅混合型甲烷單氧化酵素仍然可以被氧氣活化,但是由於部分“電子傳遞銅離子簇”被鋅離子取代,所以此酵素將電子傳遞至“基質催化銅離子簇”的能力亦隨之縮減。這些實驗亦強烈證實了銅離子在酵素中扮演電子傳遞與基質催化的角色。
Copper ions play an essential role in particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath). The particulate methane monooxygenase (pMMO) contains 15 reduced copper ions which are arranged in five trinuclear clusters. Two of these clusters were subsequently found to participate in dioxygen chemistry and hydrocarbon hydroxylation chemistry, called C-clusters. The remaining copper ions were in the reduced d10 state, and were thought to be responsible for channeling electrons to the C-clusters from NADH, called E-clusters. The low temperature EPR spectrum of as-isolated pMMO was deconvoluted into a type 2 Cu(II) signal and a broad, but nearly isotropic EPR signal centered at g ~ 2.1. Earlier magnetization and magnetic susceptibility measurements have suggested that the latter EPR signal, which is not sensitive to microwave power saturation, arise from a ferromagnetically exchange-coupled trinuclear Cu(II) cluster with J = 15�{20 cm–1 and with a zero-field splitting D of +0.018cm-1 (175 G) and E value of 0.005 cm-1 (50 G). By combining EPR spectroscopy and rapid cryogenically trap, we successfully observed the different oxidative phases of the turnover cycle and practically proved the catalytic mechanisms of pMMO.
Processing cell growth in a fermentor adapted with a hollow-fiber bioreactor, we successfully prepared the (Cu, Zn)-pMMO. The bulk of the copper ions of the E-clusters have been replaced by divalent Zn ions in (Cu, Zn)-pMMO. The Cu and Zn contents in the (Zn, Cu)-pMMO were determined by both ICP-MS and x-ray absorption K-edge spectroscopy. Further characterization of the (Zn, Cu)-pMMO was provided by low temperature electron paramagnetic spectroscopy during reductive titration and hydrocarbon hydroxylation. These studies indicate that the (Zn, Cu)-pMMO is still capable of supporting the activation of dioxygen, but that the replacement of the E-cluster copper ions has compromised the ability of the protein to mediate the transfer of reducing equivalents to the C-clusters. These observations provide strong support for the electron transfer and catalytic roles that we have previously proposed for the E-cluster and C-cluster copper ions, respectively.
Table of contents

Abstract…….……………………………………..………………….…........................ i
中文摘要………………………..……………………………………………………… iii
謝誌……..……………………………………………………………………………… iv
Table of contents………………………………………………………………………. vi


Chapter 1:
Introduction……….…………………..………………………....................................... 1

1.1 Methanothrophs…………………………………..…………………………….. 2
1.2 MMOs: sMMO and pMMO………………….………..……..….……………... 3
1.3 Hydroxylation of alkanes by pMMO. Unusual regioselectivity and stereoselectivity….………......................................................................................
7
1.4 Unsettled issues……….…….………………………………………..………….. 11


Chapter 2:
The role of copper ions on pMMO……………….…………………..………….. 12

2.1 Transcriptional switch and metabolic activation….………………………...... 13
2.2 High yield expression and purification of pMMO…………...…….………….. 16
2.3 Electron transfer and catalytic activity………………………………………... 19


Chapter 3:
Electron paramagnetic resonance and X-ray absorption spectra of copper ions…………………………………………………………………………………...…… 25

3.1 Basic principles of EPR…………………..…………………………………….. 26
Introduction……………………………………………………………..……...... 26
Magnetic moments and magnetic resonance (g-values)……………….……….. 26
Hyperfine splittings………………….…………………………..………………. 29
Spectral Anisotropy……………………………….….…………..………………. 31
Spin Hamitonians……………………..………………….……………………… 33
3.2 EPR signal of Cu(II)……………………………………….……......................... 35
3.3 EPR of a trinuclear copper cluster……………………………………...……... 39
3.4 Power saturation and EPR relaxation………………………………………… 44
3.5 X-ray absorption spectroscopy of copper ions………………………………... 47


Chapter 4: Identification and characterization of a trinuclear copper cluster in pMMO…...………………………..……………...…………..…………...... 51

4.1 Introduction.………………..………………………………...……………….…. 52
4.1 Materials and methods.………………..………………….………………….…. 53
pMMO-Enriched Membranes from Methylococcus capsulatus (Bath)……….... 53
EPR spectroscopy…………………………………….……..…………..……….... 54
pMMO activity assay…………………………………………………….………... 55
EPR deconvolution……………………………………………………………….. 55
4.2 The EPR signals of pMMO-enriched membreanes…………………………… 56
Power saturation the EPR signal of pMMO-enriched membranes……………... 56
Origin of 14N superhyperfine in type 2 Cu(II) EPR……….…………………….. 58
4.3 Simulation of the EPR spectra of pMMO ……….……..……………..………. 60
4.4 Oxidative conversion of pMMO in the presence or in the absence of hydrocarbon substrate…………………………………………………………... 64
The dioxygen chemistry mediated by the C-clusters in pMMO in the absence of hydrocarbon substrate…………………..…………………………….....….......... 64
Hydrocarbon substrate binding and hydroxylation during enzyme turnover…………..………………………………….……………………………. 68
Coupling of dioxygen chemistryto alkane hydroxylation. Experiments with methane as substrate…………………………………..…………………….....… 71
Coupling dioxygen chemistry to alkane hydroxylation. Experiments with propane as substrate……………………………………..……………………….. 73
Coupling dioxygen chemistry to alkane/alkene hydroxylation. Experiments with propylene as substrate………………….………...…..................................... 75
4.5 Conclusions………………..…………………………………….………….….... 82



Chapter 5:
(Cu, Zn)-pMMO………………………………………………………………..…….... 84

5.1 Motivation for preparation of the (Cu, Zn)-pMMO ……………………….…. 85
5.2 Preparation of a (Cu, Zn)-pMMO …………………………..………………….. 85
5.3 Metal contents ………………………………………………………...……….… 86
5.4 Reduction of (Cu, Zn)-pMMO …………………….……………………………. 88
5.5 Activity of (Cu, Zn)-pMMO ………………………………….………………..... 89
5.6 Summary ………………………………………………………..………………... 91


Chapter 6:
Summary of thesis………………………………………………………...................... 93


References……………………………………………………………...…………….….. 96
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