嗜甲烷菌中微粒體甲烷單氧化酵素分布於細胞內之脂質膜中，且為一種富含銅離子之膜上蛋白質。從過去的研究顯示，其蛋白質的調控及表現、電子傳遞以及氧化反應的進行，均與銅離子有關。甲烷單加氧酵素的作用是將甲烷羥化成為甲醇。
為了瞭解活性中心的結構與環境，因此我們利用氧化還原滴定來探討電子傳遞的情形。更進一步的，從中得知銅離子所扮演的角色。另外，經由氮原子的同位素實驗，研究電子順磁光譜的訊號以瞭解其為何種原子所造成。在模擬物實驗中，我們算出其產物的結合常數，並希望從中模擬還原態甲烷單氧化酵素和三價鐵氰化合物的實驗。

Methanotrophic bacteria use methane as both a carbon and an energy source. This biological process can be harnessed for industrial use of methane as a chemical feedstock or conversion to methanol as a liquid fuel source. The pMMO isolated from Methylococcus capsulatus (Bath) consists of a three-subunit hydroxylase (45, 27 and 23 kDa) and a NADH oxidoreductase (38 kDa). The hydroxylase is a copper protein, with 15 copper ions arranged in five trinuclear copper clusters.
In order to explore the structure of the active site as well as the nature of the reaction intermediate(s) formed at the active site during turnover of the enzyme, we have subjected the pMMO to different levels of reductants such as dithionite, as well as oxidants, including ferricyanide.

Both the catalytic clusters (C-cluster) and electron transfer sites were examined by EPR spectroscopy to distinguish between various multi-oxidation states of the copper clusters. From the disappearance and the presence of the type 2 Cu(II) EPR signals and multiline pattern with incremental addition of reductive as well as oxidative reagents, we have been able to distinguish between the two sites from the titration experiments. With the above results, we have made an important step forward in our efforts toward defining the functional role of some of the copper ions in the catalytic pathway of the pMMO.

From the EPR spectrum of the as-isolated copper enriched particulate methane monooxygenase, we have observed a normal type 2 Cu(II) EPR signal with g//=2.24, A//=185G, and g^=2.058 associated with hyperfine splitting of about 150 G centered at g//=2.19. In 15N-labeling pMMO experiments, the multiline pattern observed may be assigned to a catalytic center coupled with nitrogen atoms.

In UV-Visible experiments, copper nitrate reacted with potassium ferrocyanide and potassium ferricyanide, respectively. We have measured the binding constants of Cu-ferrocyanide and Cu-ferricyanide adducts. Unfortunately, we did not find the absorbance bands of Cu-ferrocyanide or Cu-ferricyanide adducts by the fully reduced pMMO reacted with potassium ferricyanide.

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