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。甲烷單氧化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，氘氚乙烷的實驗中，可以發現所有的氘氚乙醇都是完全的保留在反應前的立體組態，由此可以推測此酵素的反應是走向同步化反應中間體的反應機構。然而，在利用正丙烷, 正丁烷, 正戊烷來探討此酵素的反應機構時，卻顯現更為複雜的立體選擇性，其選擇性乃是基於活性中心反應空間的限制，使得活性中心在趨向pro-R或pro-S的碳氫鍵氧化反應時，呈現兩種不同的組態，而不具較高的光學活性。為了更清楚的了解烷分子與甲烷單氧化酵素之間的交互作用，我們則擴展至一系列含氘丁烷，包含[2-2H2]butane, [1-2H3]-, [1-2H3,4-2H3]-butane及 d,l form [2-2H,3-2H]butane等，將所獲得的結果做更進一步的討論。在[2-2H2]butane的實驗中，我們得到等量的(2R)-[3-2H2]butan-2-ol 及 (2R)-[2-2H]butan-2-ol產物分布，同時，當氧化發生在第二號位碳上，其立體選擇性則是趨向於單一立體組態，但從分析各項產物的分布來結論各個含氘丁烷的反應性，顯示當一級碳或二級碳所有的氫原子被氘原子所取代時，其反應性則呈現顯著的加快，表示分子的形狀或凡得瓦半徑，會因為氘原子的取代而有了顯著的變化，進而影響了Michaelis-Menten 動力學中KM的降低。另一方面，在具備光學活性的d,l型態的丁烷異構物的產物分析結果□，由於氧化過程的進行方式已直接由氫氘動力學效應來決定，產物的分佈，即直接顯現丁烷異構物在活性中心的立體位向，而少至可忽略不計的反向產物的生成，則更進一步證明了在丁烷的實驗結果與乙烷是一致的，均是直接保持原來的立體組態，也就是說，甲烷單氧化酵素的羥化反應機構是由側面不改變立體組態的情況下，將”oxene“氧原子直接嵌入碳氫鍵羰羥化就如同一般的亞碳基的嵌入反應。

Experiments on chiral ethanes have indicated that the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) catalyzes the hydroxylation of ethane with total retention of configuration. It seem to rule out a radical mechanism for the hydroxylation chemistry, at least as mediated by this enzyme. The interpretation of subsequent experiments on n-propane, n-butane and n-pentane has been complicated by hydroxylation at both the pro-R and pro-S secondary “C-H” bonds. It has been suggested that these results merely reflect presentation of both the pro-R and pro-S “C-H” bonds to the hot “oxygen atom” species generated at the active site, and that the oxo-transfer chemistry, in fact, proceeds concertedly with retention of configuration. Now, we have augmented these earlier studies with experiments on [2-2H2]-, [1-2H3]-, [1-2H3,4-2H3]-butane and designed d,l form chiral deuterated butanes. Essentially equal amounts of (2R)-[3-2H2]butan-2-ol and (2R)-[2-2H]butan-2-ol are produced upon hydroxylation of [2-2H2]butane. The chemistry is regiospecific with full retention of configuration at the secondary carbon oxidized. Moreover, deuterium enriched in primary or secondary carbon of butane molecules presented higher hydroxylation reactivity due to the reduction of the van der Waal radius of the deuterium replacement resulting in the decrease of the parameter kM deduced from Michaelis-Menten Kinetics. In the case of the various chiral deuterated butanes, the distribution of products mirrors the stereochemical configurations of the chiral butane substrates, after allowance is made for the expected deuterium isotope effect on the kinetics of the “oxygen atom” insertion step. The extent of configurational inversion has been shown to be negligible for all the chiral butanes examined. Thus, the hydroxylation of butane takes place with full retention of configuration in butane as well as in the case of ethane. These results are interpreted in terms of an oxo-transfer mechanism based on side-on singlet “oxene” insertion across the “C-H” bond similar to that previously noted for singlet carbene insertion.

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