Muthusamy Mylrajan, Ambundo Edna A, George Simon J, Lippard Stephen J, Thorneley Roger N F
Department of Biological Chemistry, John Innes Centre, Norwich, NR4 7UH, UK.
J Am Chem Soc. 2003 Sep 17;125(37):11150-1. doi: 10.1021/ja036081r.
The hydroxylase component (MMOH) of soluble methane monooxygenase from Methylococcus capsulatus (Bath) was reduced to the diiron(II) form and then allowed to react with dioxygen to generate the diiron(IV) intermediate Q in the first phase of a double-mixing stopped-flow experiment. CD3NO2 was then introduced in the second phase of the experiment, which was carried out in D2O at 25 degrees C. The kinetics of the reaction of the substrate with Q were monitored by stopped-flow Fourier transform infrared spectroscopy, observing the disappearance of the asymmetric NO2 bending vibration at 1548 cm-1. The data were fit to a single-exponential function, which yielded a kobs of 0.45 +/- 0.07 s-1. This result is in quantitative agreement with a kobs of 0.39 +/- 0.01 s-1 obtained by observing the disappearance of Q by double-mixing stopped-flow optical spectroscopy at its absorption maximum of 420 nm. These results provide for the first time direct monitoring of the hydroxylation of a methane-derived substrate in the MMOH reaction pathway and demonstrate that Q decay occurs concomitantly with substrate consumption.
在双混合停流实验的第一阶段，来自荚膜甲基球菌（巴斯）的可溶性甲烷单加氧酶的羟化酶组分（MMOH）被还原为二价铁（II）形式，然后与双原子氧反应生成二价铁（IV）中间体Q。在实验的第二阶段引入CD3NO2，该实验在25℃的重水中进行。通过停流傅里叶变换红外光谱监测底物与Q反应的动力学，观察1548 cm-1处不对称NO2弯曲振动的消失。数据拟合为单指数函数，得到的表观速率常数kobs为0.45±0.07 s-1。该结果与通过在420 nm吸收最大值处用双混合停流光光谱观察Q的消失所获得的0.39±0.01 s-1的表观速率常数在数量上一致。这些结果首次直接监测了MMOH反应途径中甲烷衍生底物的羟化作用，并证明Q的衰减与底物消耗同时发生。
