中 文 摘 要
　　Methylococcus capsulatus (Bath)為嗜甲烷菌的一種，隨著環境中銅離子濃度的高低可以調控其分別表現不同型態的微粒型甲烷單加氧酶(pMMO)及可溶型甲烷單加氧酶(sMMO)。pMMO 為膜蛋白相對於在純化上以及了解其催化機制也較為困難。過去十幾年來利用電子順磁共振光譜儀以及X光吸收光譜等實驗證實了pMMO為含有多個銅離子的蛋白質(約為12~15 個)，這些銅離子可分為C-clusters 以及E-clusters 兩大類，其中E-clusters位於PmoB單元上扮演著傳遞電子至C-clusters進行催化反應的角色，因此探討銅離子與該暴露水層蛋白質間親和鍵結能力或是配位是很重要的議題。本篇論文主要是以基因工程的方法表達PmoB 單元蛋白質，並觀察在不同氧化態銅離子的環境下這些部分表達蛋白質的摺疊構形狀況以及銅離子與蛋白質間的作用關係，透過原子吸收光譜進行銅離子定量進而利用Scatchard plot 分析暴露水層蛋白質對銅離子的鍵結常數以及最大鍵結銅離子數目。由實驗結果發現銅離子扮演幫助穩定蛋白質摺疊以及與蛋白質配位兩種重要的角色，並且得知PmoB 單元暴露於水層蛋白質所含銅離子最大鍵結數目約為16.70 個。未來研究將著重於純化其它片段重組蛋白質，希望更進一步了解暴露水層蛋白質的哪些區域對於銅離子有較強的結合作用，並且將純化後之重組片段蛋白質進行蛋白質結晶進而解析其結構並偵測晶體內配位銅離子的含量。

　　Methylococcus capsulatus (Bath) is one of methanotrophic bacteria.Differential expression of either sMMO or pMMO is regulated by the concentration of copper ions available to the cells. During the past decade, the experiments from either electron paramagnetic resonance (EPR) or X-ray absorption spectroscopy conclude that pMMO is a multicopper protein. The copper ions in the pMMO were classified into C-clusters and E-clusters. The E-clusters found to be associated with water-exposed domains of the 45 kDa subunit which were responsible for channeling electrons to the C-cluster. It would be crucial to shed light on the copper binding affinity on even coordination feature in this
aqueous domain of PmoB subunit to show how the electrons transfered from E-Cluster to C-cluster where the methane oxidation take place. Therefore, over-expressed PmoB subunit, a water-soluble domain of of pMMO in E.coli. system was conducted by using gene engineering technique. Thus, whether partially expressed proteins can be folded properly or the copper ions exhibt capability to sequester can be possilbly deduced. Furthermore, the copper concentration was quantitated with atomic absorption spectroscopy, and the copper binding constant was examessed by Scatchard plot. The results indicated that the copper ion played at least two important roles in both coordinating with protein and stablized the protein folding structure. The maximum number of protein binding copper is about 16.70. In the future, we will express other domains of water-exposed protein to study which sequence has stronger copper binding effect, and resolve their structures and copper coordinations via X-ray crystallography.

1. Amaral, J. A., Archambault, C., Richards, S. R. and Knowles, R.,
Denitrification associated with Groups I and II methanotrophs in a gradient
enrichment system. FEMS microbiol. ecol. 1995, 18, 289-298.
2. Bartle, E., Exploiting a gaseating bacteria. The Department of Molecular
Biology at the University of Bergen (UiB) 2002,
www.uib.no/elin/elpub/uibmag/en02/bacteria.html.
3. Beilen, J. B. V., Li, Z., Duetz, W.A., Smits, T.H.M. and Witholt, B.,
Diversity of Alkane Hydroxylase Systems in the Environment. Oil & Gas
Science and Technology 2003, 58, (4), 427-440.
4. Chan, S. I., Chen, K. H. C., Yu, S. S. F., Chen, C. L. and Kuo, S. S.
J., Toward Delineating the Structure and Function of the Particulate
Methane Monooxygenase from Methanotrophic Bacteria. Biochemistry
2004, 43, 4421-4430.
5. Chen, C. L., Chen, H. C., Ke, S. C.,Yu, S. F. and Chan, S. I. ,
Preparation and characterization of a (Cu,Zn)-pMMO from Methylococcus
capsulatus (Bath). J. Inorg. Biochem. 2004, 98, 2125–2130.
6. Chistoserdova, L., Vorholt,J. A. and Lidstrom M. E., A genomic
view of methane oxidation by aerobic bacteria and anaerobic archaea.
Genome Biology 2005, 6, (2), 208-214.
7. Dr. Cindy Van Doverco-Chief Scientist and Marjorie S. Curtis
Associate Professor College of William and Mary
8. Elliott, S. J., Zhu, M., Tso, L., Nguyen, H. H.T., Yip, J. H. K. and
Chan, S. I., Regio- and Stereoselectivity of Particulate Methane
Monooxygenase from Methylococcus capsulatus (Bath). J. Am. Chem. Soc.
1997, 119, 9949-9955.
9. Fjellbirkeland, A., Kleivdal H., Joergensen, C., Thestrup, H. and
Jensen, H. B., Outer membrane proteins of Methylococcus
capsulatus(Bath). Arch. Microbiol. 1997, 168, 128-135.
10. Gilbert, B., Mcdonald, I. R., FINCH, R., Stafford, G. P., Nielsen, A.
K., and Murrell, J. C., Molecular Analysis of the pmo (Particulate
Methane Monooxygenase)Operons from Two Type II Methanotrophs. Appl.
Environ. Microbiol. 2000, 66, 966.
11. Green, J., Prior S. D. and Dalton, H., Copper ions as inhibitors of
protein C of soluble methane monooxygenase of Methylococcus capsulatus
(Bath). Fur. J. Biochem. 1985, 153, 137-144.
12. Hanson, R. S. and Hanson, T. E., Methanotrophic Bacteria. Microbiol.
Rev. 1996, 60, (2), 439–471.
13. Kao, W. C., Chen, Y. R., Yi, E. C., Lee, H., Tian, Q., Wu, K. M., Tsai.
S. F.,Yu, S. S. F., Chen,Y. J., Aebersold, R. and Chan, S. I.,
Quantitative Proteomic Analysis of Metabolic Regulation by Copper Ions
in Methylococcus capsulatus (Bath). J. Biol. Chem. 2004, 279, 51554 -
51560.
14. Karlsen, O. A., Ramsevik, L., Bruseth, L. J., Øivind Larsen,
Brenner, A., Berven, F. S., Jensen, H. B. and Lillehaug, J. R.
Characterization of a prokaryotic haemerythrin from the methanotrophic
bacterium Methylococcus capsulatus (Bath). , Zoning Laws: Windows to
the Deep Chemosynthetic Relationship. FEBS J. 2005, 272, 2428-2440.
15. Labinger, J. A., Selective alkane oxidation: hot and cold approches to a
hot problem. Journal of Molecular Catalysis A: Chemical 2004, 220,
27-35.
16. Lehninger, Principle biochemistry. 439.
17. Lelieveld, J., Crutzen, P. J. and Bruühl, C., Climate Effects of
Atomosphere Methane. Chemosphere 1993, 26, (1-4), 739-768.
18. Lieberman, R. L. and Rosenzweig, A. C., Crystal structure of a
membrane-bound metalloenzyme that catalyses the biological oxidation of
methane. 2004.
19. Madigan, M. T., Martnko, J. M. and Parker, J., Prokaryote diversity:
Bacteria. Biology of Microorganisms. 8th ed. 1997, 16, 666-671.
20. Murrell, J. C., Gilbert, B. and McDonald, I. R., Molecular biology and
regulation of methane monooxygenase. Arch Microbiol 2000, 173,
325–332.
21. Murrell, J. C., McDonald, I. R. and Gilbert, B., Regulation of
expression of methane monooxygenases by copper ions. Trends in
Microbiology 2000, 8, (5), 221-225.
22. Naomi, W., Øivind Larsen.et.al., Genomic Insights into
Methanotrophy:The Complete Genome Sequence of Methylococcus
capsulatus (Bath). PLoS Biology 2004, 2, (10), e303.
23. Nguyen, H. H. T., Elliott, S. J., Yip, J. H. K. and Chan, S. I., The
Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath)
Is a Novel Copper-containing Three-subunit. J. Biol. Chem. 1998, 273,
(14), 7957–7966.
24. Nguyen, H. H. T., Nakagawa, K. H., Hedman, B., Elliott, S. J.,
Lidstrom, M. E., Hodgson, K. O. and Chan, S. I., X-ray Absorption
and EPR Studies on the Copper Ions Associated with the Particulate
Methane Monooxygenase from Methylococcus capsulatus (Bath). Cu(I)
Ions and Their Implications. J. Am. Chem. Soc. 1996, 118, 12766-12776.
25. Nguyen, H. H. T., ShiemkeSP, A. K., Jacobs, S. J., Brian J.,
Halesn, Lidstromll, M. E. and Chan, S. I., The Nature of the Copper
Ions in the Membranes Containing the Particulate Methane Monooxygenase
from Methylococcus capsulatus (Bath). J. Biol. Chem. 1994, 269, (21),
14995-15005.
26. Nielsen, A. K., Gerdes, K. and Murrell, J. C., copper-dependent
reciprocal transcriptional regulation of methane monooxygenase genes in
methylococcus capsulatus and methylosinus trichosporium. Mol. Microbiol.
1997, 25, (2), 399-409.
27. Periana, R. A., Bhalla,G., Tenn, W. J., Young , K. J. H., Liu, X. Y.;
Mironov, O., Jones C. J. and Ziatdinov, V. R., Perspectives on some
challenges and approaches for developing the next generation of selective,
low temperature, oxidation catalysts for alkane hydroxylation based on the
CH activation reaction. Journal of Molecular Catalysis A: Chemical
2004, 220, 7-25.
28. Periana, R. A., Taube, D. J., Evitt, E. R., Loffler, D. G., Wentrcek,
P. R., Voss, G. and Toshihiko Masuda, A Mercury-catalyzed,
High-Yield System for the Oxidation of Methane to Methanol. Science
1993, 259, 340-343.
29. Rapisarda, V. A., Chehn, R. N., Javier De Las Rivas, Luisa R. M.,
Faras, R. N. and Massa, E. M., Evidence for Cu(I)-thiolate ligation and
prediction of a putative copper-binding site in the Escherichia coli NADH
dehydrogenase-2. Arch. Biochem. Biophys. 2002, 405, 87–94.
30. Ricke, P., Erkel, C., Kube, M., Reinhardt, R. and Liesack, W.,
Comparative Analysis of the Conventional and Novel pmo
(ParticulateMethane Monooxygenase) Operons from Methylocystis Strain
SC2. Appl. Environ. Microbiol. 2004, 70, (5), 3055-3063.
31. Ro´ bert Csa´ ki, Bodrossy, L., Jo´ zsef Klem, Murrell, J. C. and
Korne´ l L. Kova´cs, Genes involved in the copper-dependent regulation
of soluble methane monooxygenase of Methylococcus capsulatus (Bath):
cloning,sequencing and mutational analysis. Microbiology 2003, 149,
1785-1795.
32. Semrau, J. D., Chistoserdov, A., Lebron, J., Costello, A.,
Davagnino, J., Kenna, E., Holmes, A. J., Finch, R., Murrell, J. C.
and Lidstrom, M. E., Particulate Methane Monooxygenase Genes in
Methanotrophs. 1995, 177, (11), 3071–3079.
33. Steinkamp, R., Zimmer, W. and Papen, H., Improved Method for
Detection of Methanotrophic Bacteria in Forest Soils by PCR. Cur.
Microbiol. 2001, 42, 316–322.
34. Stolyar, S., Costello, A. M., Peeples, T. L. and Lidstrom, M. E.,
Role of multiple gene copies in particulate methane monooxygenase activity
in the methane-oxidizing bacterium Methylococcus capsulatus Bath.
Microbiology 1999, 145, 1235–1244.
35. Stolyar, S., Franke, M. and Lidstrom, M. E., Expression of Individual
Copies of Methylococcus capsulatus (Bath) Particulate
MethaneMonooxygenase Genes. J. Bacteriol. 2001, 183, (5), 1810–1812.
36. Strom,T., Ferenci, T. and Quayle, J. R., The Carbon Assimilation
Pathways of Methylococcus, Psedomonas methanica and Methylosinus
trichosporium. Biochem. J. 1974, 144, 465-476.
37. Waheln, M., The global methane cycle. Annu. Rev. Earth Planet. Sci.
1993, 21, 407-426.
38. Whittembury, R., Phillps,K. C., and Wilkinson, J. F., Enrichment,
Isolation and Some Properties of Methane-utilizing Bacteria. Journal of
General Microbiology 1970, 61, 205-218.
39. Yu, S. S. F., Chen, K. H. C.,Tseng, M. Y. H., Wang,Y. S., Tseng, C.
F., Chen, Y. J., Huang, D. S. and Chan, S. I., Production of
High-Quality Particulate Methane Monooxygenase in High Yields from
Methylococcus capsulatus (Bath) with a Hollow-Fiber Membrane
Bioreactor. J. Bacteriol. 2003, 185, (20), 5915–5924.
40. Yu, S. S. F., Wu, L. Y., Chen, K. H. C. Luo, W. I., Huang, D. S. and
Chan, S. I., The Stereospecific Hydroxylation of [2,2-2H2]Butane and
Chiral Dideuteriobutanes by the Particulate Methane Monooxygenase
from Methylococcus capsulatus (Bath). J. Biol. Chem. 2003, 278, (42),
40658–40669.
41. ZAHN, J. A. and Dispirito, A. A., Membrane-Associated Methane
Monooxygenase from Methylococcus capsulatus (Bath). J. Bacteriol. 1996,
178, (4), 1018–1029.
42. 郭博堯, 全球化石能源危機時代與我國所面臨挑戰. 財團法人國家政
策研究發展基金會永續發展組研究報告 2002, 12月.
43. 郭博堯, 全球石油危機的油價衝擊. 財團法人國家政策研究發展基金
會永續發展組研究報告 2003, 1月.
44. 程人俠譯, 地球的能源. 科學月刊 1973, 7月, (43期).