Unmasking of deuterium kinetic isotope effects on the methane monooxygenase compound Q reaction by site-directed mutagenesis of component B
Unmasking of deuterium kinetic isotope effects on the methane monooxygenase compound Q reaction by site-directed mutagenesis of component B
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DOI：
10.1021/ja016632i
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发表时间：
2001-10-24
影响因子：
15
通讯作者：
Lipscomb, JD
中科院分区：
文献类型：
--
作者：
Brazeau, BJ;Wallar, BJ;Lipscomb, JD
甲烷氧化菌（例如毛孢甲基红菌 OB3b）利用甲烷作为碳和能量的唯一来源。 1 甲烷代谢的第一步是在甲烷单加氧酶 (MMO) 的催化下，O2 偶联氧化成甲醇。可溶性 MMO 系统由三个组件组成：羟化酶 (MMOH)、还原酶 (MMOR) 和称为 MMOB 的小效应蛋白。 2 该酶的活性位点位于 MMOH 中，包含一个双核铁簇，该簇埋藏在 MMOH 表面以下至少 12 Å 处，没有通向本体溶剂的通道。 3 MMOB 已被证明在 MMO 催化的多个方面具有效应器作用。 4 特别是，我们的研究表明，MMOB 可将 O2 与不同的 MMOH 簇之间的反应速率加快 1000 倍，从而启动催化循环。 5 该循环中的后期中间体称为 Q，已被证明是生物学中最强大的氧化剂之一。 6 它包含一个双-μ-oxo-Fe (IV) 2 簇7，可直接与底物发生反应。 Q 的黄色发色团允许其形成并随后与底物发生反应，然后采用停流技术。 MMO系统是化学中为数不多的案例之一，也是生物学中唯一可以直接观察到氧插入未活化CH键的反应的案例之一。我们最近的研究表明，Q 和底物 (S) 之间的反应分两步发生： 8 其中 kSB 是 S 结合和解离速率常数的函数，kCH 是 CH 键断裂速率常数。速率决定步骤显然取决于底物的大小和要裂解的 CH 键的强度。该模型已用于解释以下值得注意的现象：Q 与甲烷的反应具有约 50, 9 的初级氘 KIE，而与乙烷的相同反应则表现出统一的 KIE。我们假设，对于甲烷，CH 键断裂是限速步骤，10 而乙烷较弱的 CH 键的更快裂解及其较大的尺寸使得结合成为限速步骤，从而掩盖了同位素效应。因此，观察到乙烷的产物同位素效应为 4.2，11 表明反应中存在同位素敏感步骤，即使它不是 Q 的单周转反应的速率限制。尽管 MMOB 的 O2 门控效应已得到充分记录，但直到最近才知道它也会影响整个催化循环的中间转化率。受其 NMR 溶液结构启发，MMOB 的定点诱变现已被用于表明催化循环的几个步骤，包括 Q 反应，受到该组分的显着影响。一个突变体 N107G/S109A/S110A/T111A (MMOBquad) 13 产生一种 MMOB 形式，可加速与大底物（例如呋喃）的 Q 衰变反应速率，并降低与最小底物（甲烷）的反应速率。基于这些结果，我们提出 MMOB 最普遍的功能是打开一个进入 MMOH 活性位点的通道，该通道根据 O2 和 CH4 的大小进行调整，以便 MMOH 充当该大小分子的分子筛。综合考虑这两项研究，如果未能观察到大底物的 KIE 是由于结合缓慢，而 MMOBquad 加速结合，则在单次周转期间使用 MMOBquad 代替 MMOBwt 时，较大的底物可能会表现出同位素效应，这似乎是合理的。在这项研究中，我们研究了 Q 与甲烷、乙烷和丙烷之间反应的反应速率和氘 KIE。结果与 MMOB 的模型一致……
Methanotrophs such as Methylosinus trichosporium OB3b utilize methane as the sole source of carbon and energy. 1 The first step in the metabolism of methane is the O2 coupled oxidation to methanol which is catalyzed by methane monooxygenase (MMO). The soluble MMO system consists of three components, a hydroxylase (MMOH), a reductase (MMOR), and a small effector protein termed MMOB. 2 The active site of the enzyme, located in MMOH, contains a binuclear iron cluster that is buried at least 12 Å below the surface of MMOH with no access channel to bulk solvent. 3 MMOB has been shown to have an effector role in several aspects of MMO catalysis. 4 In particular, our studies have shown that MMOB accelerates the rate of the reaction between O2 and the diferrous MMOH cluster by 1000-fold to initiate the catalytic cycle. 5 A later intermediate in the cycle, termed Q, has been shown to be among the most powerful oxidants in biology. 6 It contains a bis-μ-oxo-Fe (IV) 2 cluster7 that reacts directly with substrates. The yellow chromophore of Q allows its formation and subsequent reaction with substrates to be followed by stopped-flow techniques. The MMO system represents one of the few cases in chemistry and the only case in biology in which the oxygen insertion reaction into unactivated CH bonds can be directly observed. Our recent studies suggest that the reaction between Q and substrates (S) occurs in two steps: 8 where kSB is a function of the S binding on and off rate constants and kCH is the CH bond breaking rate constant. The rate-determining step apparently depends on the size of the substrate and the strength of the CH bond to be cleaved. This model has been used to account for the remarkable observation that the reaction of Q with methane has a primary deuterium KIE of about 50, 9 while the same reaction with ethane exhibits a KIE of unity. We hypothesize that for methane, CH bond-breaking is rate-limiting, 10 whereas the combination of faster cleavage of the weaker CH bond of ethane and its larger size makes binding the rate-limiting step, thereby masking the isotope effect. Accordingly, a product isotope effect of 4.2 is observed for ethane, 11 showing that there is an isotope sensitive step in the reaction even though it is not rate-limiting for the single-turnover reaction with Q.Although the O2 gating effect of MMOB has been well documented, it was not known until recently that it also affects rates of intermediate conversion throughout the catalytic cycle. Site-directed mutagenesis of MMOB inspired by its NMR solution structure12 has now been used to show that several steps of the catalytic cycle, including the Q reaction, are significantly affected by this component. One mutant N107G/S109A/S110A/T111A (MMOBquad) 13 resulted in a form of MMOB that accelerates the rate of the Q decay reaction with large substrates (eg, furan) and decreases the reaction rate with the smallest substrate, methane. On the basis of these results, we proposed that the most generalized function of MMOB is to open a channel into the MMOH active site that is tuned to the size of O2 and CH4 so that MMOH acts as a molecular sieve for this size molecule. Considering these two studies together, it seems reasonable that if the failure to observe KIEs for large substrates is due to slow binding and if MMOBquad accelerates binding, then larger substrates may exhibit an isotope effect when MMOBquad is used in place of MMOBwt during a single turnover. In this study, we have investigated reaction rates and the deuterium KIEs for the reactions between Q and methane, ethane, and propane. The results are in agreement with the model in which MMOB …