Intermediate Q from soluble methane monooxygenase hydroxylates the mechanistic substrate probe norcarane: Evidence for a stepwise reaction
Intermediate Q from soluble methane monooxygenase hydroxylates the mechanistic substrate probe norcarane: Evidence for a stepwise reaction
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DOI：
10.1021/ja016376
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发表时间：
2001-12-05
影响因子：
15
通讯作者：
Lipscomb, JD
中科院分区：
文献类型：
--
作者：
Brazeau, BJ;Austin, RN;Lipscomb, JD
降冰片烷是一种有价值的酶催化烃氧化反应的机理探针，因为不同的产品或产品分布的协同，自由基，和阳离子为基础的反应的结果。来自发孢甲基弯孢菌（Methylosinus trichosporium）OB 3b的可溶性甲烷单加氧酶（sMMO）催化降冰片烷氧化生成3-羟甲基环己烯和3-环庚烯醇，除了2-和3-降冰片烷醇之外，它们分别是自由基和阳离子中间体的特征化合物。过去的单营业额瞬态动力学研究已经确定了几个光学不同的中间体从催化循环的羟化酶组分的sMMO。因此，可以直接监测降冰片烷和关键反应中间体之间的反应。降冰片烷的存在只增加了一种中间体的衰变速率，即高价的含双-μ-氧代Fe（IV）（2）簇的化合物Q，表明它负责大部分的氧化化学。从自由基和阳离子的中间体从降冰片烷氧化sMMO催化的产物的观察是一致的，其中初始底物自由基中间体形成的氢原子提取的机制。该中间体然后经历氧反弹、分子内重排随后氧反弹或损失第二个电子以产生OH-转移到其上的阳离子中间体。推定的自由基中间体的寿命的估计下限为20 ps是在雅阁从以前的空间位阻sMMO探针的研究确定的值。
Norcarane is a valuable mechanistic probe for enzyme-catalyzed hydrocarbon oxidation reactions because different products or product distributions result from concerted, radical, and cation based reactions. Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b catalyzes the oxidation of norcarane to afford 3-hydroxymethylcyclohexene and 3-cycloheptenol, compounds characteristic of radical and cationic intermediates, respectively, in addition to 2- and 3-norcaranols. Past single turnover transient kinetic studies have identified several optically distinct intermediates from the catalytic cycle of the hydroxylase component of sMMO. Thus, the reaction between norcarane and key reaction intermediates can be directly monitored. The presence of norcarane increases the rate of decay of only one intermediate, the high-valent bis-mu -oxo Fe(IV)(2) cluster-containing species compound Q, showing that it is responsible for the majority of the oxidation chemistry. The observation of products from both radical and cationic intermediates from norcarane oxidation catalyzed by sMMO is consistent with a mechanism in which an initial substrate radical intermediate is formed by hydrogen atom abstraction. This intermediate then undergoes either oxygen rebound, intramolecular rearrangement followed by oxygen rebound, or loss of a second electron to yield a cationic intermediate to which OH- is transferred. The estimated lower limit of 20 ps for the lifetime of the putative radical intermediate is in accord with values determined from previous studies of sterically hindered sMMO probes.