Oxygen activation catalyzed by methane monooxygenase hydroxylase component: Proton delivery during the O-O bond cleavage steps
Oxygen activation catalyzed by methane monooxygenase hydroxylase component: Proton delivery during the O-O bond cleavage steps
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DOI：
10.1021/bi982712w
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发表时间：
1999-04-06
期刊：
影响因子：
2.9
通讯作者：
Lipscomb, JD
中科院分区：
文献类型：
--
作者：
Lee, SY;Lipscomb, JD
研究了溶剂pH和氘化对三磷酸甲基藻OB3b中甲烷单加氧酶（MMO）可溶形式催化双氧活化过程中关键中间体瞬态动力学的影响。MMO由羟化酶（MMOH）、还原酶和“B”（MMOB）组成。MMOH含有羧酸盐和氧桥接双核铁簇，催化O-2活化和插入化学。二亚铁MMOH-MMOB配合物与O-2反应生成二亚铁中间化合物O (O)，随后生成二亚铁中间化合物P (P)，推测为过氧加合物。当pH值为7.7时，O衰变反应在误差范围内与pH无关(k(obs) = 22 +/- 2s (-1))；k（obs) = 26 +/- 2s (-1), pH 7.0）。相反，P的形成速率随着pH的增加而急剧下降，在pH 8.6时接近于零；观察到的速率常数适合于单次去质子化事件，pK(a) = 7.6，在4℃时k(p) = 9.1 +/- 0.9 s（-1）的最大形成速率达到pH 6.5附近。P的形成比O的消失要慢，这表明在这两者之间至少会形成一种未被检测到的中间产物（P*）。P自发地衰变成高度显色的中间产物，化合物Q (Q)。P的衰减速率与Q的生成速率一致，且随着pH的增加，P和Q的衰减速率急剧下降，在pH 8.6时接近于零；观察到的速率常数适合于单次去质子化事件，pK(a) = 7.6，在4℃时k(Q) = 2.6 +/- 0.1 s（-1）的最大形成速率达到pH 6.5附近。P的生成和衰减速率以及Q的生成速率随着反应混合物中D2O摩尔分数的增加而线性降低。在5℃时观察到k(H)/k(D) = 1.3 +/- 0.1 （P生成）和k(H)/k(D) = 1.4 +/- 0.1 （P衰变和Q生成）的动力学溶剂同位素效应值。质子库图的线性表明，在生成反应的过渡态中，每个中间体只转移了一个质子。如果这些质子被转移到结合的氧分子上，正如反应化学计量学所要求的那样，那么数据就与一个模型相一致，在这个模型中，水的形成与反应性的双氧双核铁（IV） Q的形成同时进行。
The effects of solvent pH and deuteration on the transient kinetics of the key intermediates of the dioxygen activation process catalyzed by the soluble form of methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b have been studied. MMO consists of hydroxylase (MMOH), reductase, and "B" (MMOB) components. MMOH contains a carboxylate- and oxygen-bridged binuclear iron cluster that catalyzes O-2 activation and insertion chemistry. The diferrous MMOH-MMOB complex reacts with O-2 to form a diferrous intermediate compound O (O) and subsequently a diferric intermediate compound P (P), presumed to be a peroxy adduct. The O decay reaction was found to be pH-independent within error at 4 degrees C (k(obs) = 22 +/- 2 s(-1) at pH 7.7; k(obs) = 26 +/- 2 s(-1) at pH 7.0). In contrast, the P formation rate was found to decrease sharply with increasing pH to near zero at pH 8.6; the observed rate constants fit to a single deprotonation event with a pK(a) = 7.6 and a maximal formation rate at 4 degrees C of k(p) = 9.1 +/- 0.9 s(-1) achieved near pH 6.5. The formation of P was slower than the disappearance of O, indicating that at least one other undetected intermediate (P*) must form in between. P decays spontaneously to the highly chromophoric intermediate, compound Q (Q). The decay rate of P matched the formation rate of Q, and both rates decreased sharply with increasing pH to near zero at pH 8.6; the observed rate constants fit to a single deprotonation event with a pK(a) = 7.6 and a maximal formation rate at 4 degrees C of k(Q) = 2.6 +/- 0.1 s(-1) achieved near pH 6.5. No pH dependence was observed for the decay of Q. The formation and decay rates of P and the formation rate of Q decreased linearly with mole fraction of D2O in the reaction mixture. Kinetic solvent isotope effect values of k(H)/k(D) = 1.3 +/- 0.1 (P formation) and k(H)/k(D) = 1.4 +/- 0.1 (P decay and Q formation) were observed at 5 degrees C. The linearity of the proton inventory plots suggests that only a single proton is transferred in the transition state of the formation reaction for each intermediate. If these protons are transferred to the bound oxygen molecule, as formally required by the reaction stoichiometry, the data are consistent with a model in which water is formed concurrently with the formation of the reactive bis mu-oxo-binuclear Fe(IV) species, Q.