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 The bulk of the copper ions of the E-clusters have been replaced by divalent Zn ions in (Cu, Zn)-pMMO.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.

1. Hanson, R. S.; Hanson, T. E. Micorbiol. Rev. 1996, 60, 439□471.
2. Anthony, C. In The Biochemistry of Methylotrophs; Academic Press: London, 1982.
3. Higgins, I. J.; Best, D. J.; Hammond, R. C. Nature 1980, 286, 561□564.
4. Hanson, R. S.; Netrusov, A. I.; Tsuji, K. In The Prokaryotes; Balows, A.; Truper, H. G.; Dworkin, M.; Harder, W.; Schleifer, K.-H. Eds.; Springer-Verlag: New York, 1992, Vol. I, Chap. 118.
5. Voss, D. Technol. Rev. 2002, 105, 69–72.
6. Periana, R. A.; Taube, D. J.; Gamble, S.; Taube, H.; Satoh, T.; Fujii, H. Science 1998, 280, 560–564.
7. Periana, R. A.; Taube, D. J.; Evitt, E. R.; Löffler, D. G.; Wentrcek, P. R.; Voss, G.; Masuda, T. Science 1993, 259, 340–343.
8. Murrell, J. C.; McDonald, I. R.; Gilbert, B. Trends Microbiol. 2000, 8, 221–225.
9. Hyman, M. R.; Murton, I. B.; Arp, D. J. Appl. Environ. Microbiol. 1988, 54, 3187–3190.
10. Ortiz de Montellano, P. R. In Cytochrome P450: Structure, Mechanism, and Biochemistry; Plenum: New York, 1986.
11. Colby, J.; Stirling, D. I.; Dalton, H. Biochem. J. 1977, 165, 395□402.
12. Dalton, H. Adv. Appl. Microbiol. 1980, 26, 71□87.
13. Green, J.; Dalton, H. J. Biol. Chem. 1989, 264, 17698□□7703.
14. Fox, B. G.; Borneman, J. G.; Wackett, L. P.; Lipscomb, J. D. Biochemistry 1990, 29, 6419□6427.
15. Rataj, M. J.; Kauth, J. E.; Donnelly, M. I. J. Biol. Chem. 1991, 266, 18684□18690.
16. Andersson, K. K.; Froland, W. A.; Lee, S.-K.; Lipscomb, J. D. New J. Chem. 1991, 15, 411□415.
17. Fox, B. G.; Froland, W. A.; Dege, J. E.; Lipscomb, J. D. J. Biol. Chem. 1989, 264, 10023□10033.
18. Colby, J.; Dalton, H. Biochem. J. 1978, 171, 461□468.
19. Woodland, M. P.; Dalton, H. J. Biol. Chem. 1984, 259, 53□59.
20. Gassner, G. T.; Lippard, S. J. Biochemistry 1999, 38, 12768□12785.
21. Rosenzweig, A. C.; Frederick, C. A.; Lippard, S. J.; Nordlund, P. Nature 1993, 366, 537□543.
22. Rosenzweig, A. C.; Nordlund, P.; Takahara, P.; M. Frederick, C. A.; Lippard, S. J. Chem. Biol. 1995, 2, 409□418.
23. Elango, N.; Radhakrishnan, R.; Froland, W. A.; Wallar, B. J.; Earhart, C. A.; Lipscomb, J. D.; Ohlendorf, D. H. Protein Sci. 1997, 6, 556□568.
24. Chang, S.-L.; Wallar, B. J.; Lipscomb, J. D.; Mayo, K. H. Biochemistry 1999, 38, 5799□5812.
25. Walters, K. J.; Gassner, G. T.; Lippard, S. J.; Wagner, G. Proc. Natl. Acad. Sci. USA 1999, 96, 7877□7882.
26. Whittington, D. A.; Lippard, S. J. J. Am. Chem. Soc. 2001, 123, 827□838.
27. Merkx, M.; Kopp, D. A.; Sazinsky, M. H.; Blazyk, J. L.; Műller, J.; Lippard, S. J. Angew. Chem. Int. Ed. Engl. 2001, 40, 2782□2807.
28. Stainthorpe, A. C.; Lees, V.; Salmond, G. P. C.; Dalton, H.; Murrell, J. C. Gene 1990, 91, 27□34.
29. Coufal, D. E.; Blazyk, J. L.; Whittington, D. A.; Wu, W. W.; Rosenzweig, A. C.; Lippard, S. J. Eur. J. Biochem. 2000, 267, 2174□2185.
30. Cardy, D. L. N.; Laidler, V.; Salmond, G. P. C.; Murrell, J. C. Arch. Microbiol. 1991, 156, 477□483.
31. Cardy, D. L. N.; Laidler, V.; Salmond, G. P. C.; Murrell, J. C. Mol. Microbiol. 1991, 5, 335□342.
32. McDonald, I. R.; Uchiyama, H.; Kambe, S.; Yagi, O.; Murrell, J. C. Appl. Environ. Microbiol. 1997, 63, 1898□1904.
33. Grosse, S.; Laramee, L.; Wendlandt, K.-D.; McDonald, I. R.; Miguez, C. B.; Kleber, H.-P. Appl. Environ. Microbiol. 1999, 65, 3929□3935.
34. Shigematsu, T.; Hanada, S.; Eguchi, M.; Kamagata, Y.; Kanagawa, T.; Kurane, R. Appl. Environ. Microbiol. 1999, 65, 5198□5206.
35. Nielsen, A. K.; Gerdes, K.; Degn, H.; Murrell, J. C. Microbiology 1996, 142, 1289□1296.
36. Nielsen, A. K.; Gerdes, K.; Murrell, J. C. Mol. Microbiol. 1997, 25, 399□409.
37. Chan, S. I.; Nguyen, H.-H. T.; Shiemke, A. K.; Lidstrom, M. E. In Bioinorganic Chemistry of Copper; Karlin, K. D., Tyeklar, Z., Eds.; Chapman and Hall: New York, 1993.
38. Chan, S. I.; Nguyen, H.-H. T.; Shiemke, A. K.; Lidstrom, M. E. In Microbial Growth on C1 Compounds; Murrell, J. C., Kelly, D. P., Eds.; Intercept: Andover, Hampshire, U.K., 1993.
39. Semrau, J. D.; Chistoserdov, A.; Lebron, J.; Costello, A.; Davagnino, J.; Kenna, E.; Holmes, A. J.; Finch, R.; Murrell, J. C.; Lidstorm, M. E. Journal of Bacteriology 1995, 177, 3071□3079.
40. Nguyen, H.-H. T.; Shiemke, A. K.; Jacobs, S. J.; Hales, B. J.; Lidstrom, M. E.; Chan, S. I. J. Biol. Chem. 1994, 269, 14995□15005.
41. Nguyen, H.-H. T.; Elliott, S. J.; Yip, J. H.-K.; Chan, S. I. J. Biol. Chem. 1998, 273, 7957-7966.
42. Stolyar, S.; Costello, A. M.; Peeples, T. L.; Lidstrom, M. E. Microbiology 1999, 145, 1235□1244.
43. Semrau, J. D.; Zolandz, D.; Lidstrom, M. E.; Chan, S. I. J. Inorg. Biochem. 1995, 58, 235□244.
44. Chan, S. I.; Nguyen, H.-H. T.; Shiemke. A. K.; Lidstrom, M. E. In Studies Toward Characterization of Methane Monooxygenase; Murrel, J. C.; Kelly, D. P., Eds.; Intercept: Andover, Hampshire, U.K., 1993, pp 93□107.
45. Nguyen, H.-H. T.; Nakagawa, K. H.; Hedman, B.; Elliott, S. J.; Lidstrom, M. E.; Hodgson, K. O.; Chan, S. I. J. Am. Chem. Soc. 1996, 118, 12766□12776.
46. Chan, S. I.; Chen, K. H.-C.; Yu, S. S.-F.; Chen, C.-L.; Kuo, S. S.-J., Biochemistry. 2004, 43, 4421□4430.
47. Elliott, S. J.; Zhu, M.; Tso, L.; Nguyen, H.-H. T.; Yip, J. H.-K.; Chan, S. I. J. Am. Chem. Soc. 1997, 119, 9949□9955.
48. Yu, S, S.-F.; Wu, L.-Y.; Chen, K. H.-C.; Luo, W.-I.; Huang, D.-S.; Chan, S. I. J. Biol. Chem. 2003, 278, 40658□□□□□□.
49. Kirmse, W.; Őzkir, J. S. J. Am. Chem. Soc. 1992, 114, 7590□7591.
50. Yu, S. S.-F.; Chen, K. H.-C.; Tseng, M. Y.-H.; Tseng, C.-F.; Wang, Y.-S.; Chen, Y.-J.; Huang, D.-H.; Chan, S. I. J. Bacteriol. 2003, 185, 5915□5924.
51. Stanley, S. H.; Prior, S. D.; Leak, D. J.; Dalton, H. Biotechnol. Lett. 1983, 5, 487□492.
52. Choi, D. W.; Kunz, R. C.; Boyd, E. S.; Semrau, J. D.; Antholine, W. E.; Han, J. I.; Zahn, J. A.; Boyd, J. M.; Mora, A. M.; DiSpirito, A. A. J. Bacteriol. 2003, 185, 5755□5764.
53. Prior, S. D., and Dalton, H. J. Gen. Microbiol. 1985, 131, 155□163.
54. Madigan, M. T.; Martnko, J. M.; Parker, J. In Biology of Microorganisms, 8th ed., Simon & Schuster: New York, 1997.
55. Kao, W.-C., MSD. Dissertation, NTU, Taipei, Taiwan, 2004.
56. Smith, D. D.; Dalton, H. Eur. J. Biochem.1989, 182, 667□671.
57. Lieberman, R. L.; Shrestha, D. B.; Doan, P. E.; Hoffman, B. M.; Stemmler, T. L.; Rosenzweig, A. C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3820□3825.
58. Basu, P.; Katterle, B.; Andersson, K. K.; Dalton, H. Biochem. J. 2003, 369, 417□427.
59. Takeguchi, M.; Okura, I. Catal. SurV. Jpn. 2000, 4, 51□63.
60. Xin, J. Y.,;Cui, J. R.; Hu, X. X.; Li, S. B.; Xia, C. G.; Zhu, L. M.; Wang, Y. Q. Biochem. Biophys. Res. Commun. 2002, 295, 182□186.
61. Cook, S. A.; Shiemke, A. K. J. Inorg. Biochem.1996, 64, 273□284.
62. Holm, R. H.; Kennepohl, P.; Solomon, E. I. Chem. Rev. 1996, 96, 2239□2314.
63. Marko, I. E.; Tsukazaki, M.; Giles, P. R.; Brown, S. M.; Urch, C. J. Angew. Chem. 1997, 109, 2297; Angew. Chem. Int. Ed. Engl. 1997, 36, 2208.
64. Solomon, E. I.; Jones, P. M.; May, J. A. Chem. Rev. 1993, 93, 2623□2644.
65. Reitz, J. B.; Solomon, E. I. J. Am. Chem. Soc. 1998, 120, 11 467□11478.
66. Solomon, E. I.; Tuczek, F.; Root, D. E.; Brown, C. A. Chem. Rev. 1994, 94, 827□856.
67. Klinman, J. P. Chem. Rev. 1996, 96, 2541□2562.
68. Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Chem. Rev. 1996, 96, 2563□2605.
69. Ferguson-Miller, S.; Babcock, G. T. Chem. Rev. 1996, 96, 2889□2908.
70. Solomon, E. I.; Chen, P.; Metz, M.; Lee, S.-K.; Palmer, A. E. Angew. Chem. Int. Ed. 2001, 40, 4570□4590.
71. Elliott, S. J. Ph.D. thesis, California Institute of Technology, Pasadena, CA., 2000.
72. Solomon, E. I.; Baldwin, M. J.; Lowery, M. D.; Chem. Rev. 1992, 92, 521□542
73. Cole, J. L.; Clark, P. A.; Solomon, E. I. J. Am. Chem. Soc. 1990, 112, 9534□9548.
74. Sundaram, U. M.; Zhang, H. H.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am. Chem. Soc., 1997, 119, 12525□12540.
75. Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Chem. Rev. 1996, 96, 2563□2605.
76. Kahn, O. Angew. Chem. Int. Ed. Engl. 1985, 24, 834□850.
77. Miller, J. S.; Epstein, A. J.; Angew. Chem. Int. Ed. Engl., 1994, 33, 385□415.
78. Butcher, R. J.; O'Connor, C. J.; Sinn, E. Inorg. Chem. 1981, 20, 537□545.
79. Chaudhuri, P.; Winter, M.; DellaVedova, B. P. C.; Bill, E.; Trautwein, A.; Gehring, S.; Fleischhauer, P.; Nuber, B.; Weiss, J. Inorg. Chem. 1991, 30, 2148□2157.
80. Suh, M. P.; Han, M. Y.; Lee, J. H.; Min, K. S.; Hyeon, C. J. Am. Chem. Soc., 1998, 120, 3819□3829.
81. Bernhardt, P. V.; Sharpe, P. C. J. Chem. Soc., Dalton Trans. 1998, 1087□1088.
82. Springborg, J.; Glerup, J.; Sotofte, I. Acta Chem. Scand. 1997, 51, 832□838.
83. Bazzicalupi, C.; Bencini, A.; Bencini. A.; Bianchi, A.; Corana, F.; Fusi, V.; Giorgi, C.; Paoli, P.; Paoletti, P.; Valtancoli, B.; Zanchini, C. Inorg. Chem. 1996, 35, 5540□5548.
84. Pavlova, S. V.; Chen, K. H.-C.; Chan, S. I. J. Inorg. Biochem. 2003, 96, 210.
85. Bencini, A.; Gatteschi, D. 1990, EPR of exchange-coupled systems, Springer, Berlin.
86. DuBois, J. L.; Mukherjee, P.; Stack, T. D. P.; Hedman, B.; Solomon, E. I.; Hodgson, K. O. J. Am. Chem. Soc. 2000, 122, 5775□5787
87. Elliott, S. J.; Randall, D. W.; Britt, R. D.; Chan, S. I. J. Am. Chem. Soc. 1998, 120, 3247□3248.
88. Sroll. S., Ph.D. thesis, ETH, Zurich, 2003.
89. Hung, S.-C., Ph.D. thesis, California Institute of Technology, Pasadena, CA, 2001.
90. Vinchurkar, M. S.; Chen, K. H.-C.; Yu, S. S.-F.; Kuo, S.-J.; Chiu, H.-C.; Chien, S.-H.; Chan, S. I. Biochemistry 2004 (submitted).
91. Tseng, C.-F., MSD. Dissertation, NTHU, Hsinchu, Taiwan, 2001.
92. Lemos, S. S.; Collins, M. L. P.; Eaton, S. S.; Eaton, G. R.; Antholine, W. E. Biophys. J. 2000, 79, 1085-1094.
93. Tolman, W. B. Acc. Chem. Res. 1997, 30, 227□237.
94. Mahadevan, V.; Henson, M. J.; Solomon, E. I.; Stack, T. D. P. J. Am. Chem. Soc. 2000, 122, 10249□10250.
95. Taki, M.; Itoh, S.; Fukuzumi, S. J. Am. Chem. Soc. 2001, 123, 6203□6204.
96. This total protein concentration in the membranes was determined by the Lowry method. The pMMO concentration was estimated to be 83(1)% of the total protein by SDS-gel-analysis.
97. Lee, H. Y.; Wu, T. B.; Lee, J. F. J. Appl. Phys. 1996, 80, 2175□2180.