Stopped-flow Fourier transform infrared spectroscopy of nitromethane oxidation by the diiron(IV) intermediate of methane monooxygenase

被引:11
作者
Muthusamy, M
Ambundo, EA
George, SJ
Lippard, SJ
Thorneley, RNF
机构
[1] MIT, Dept Chem, Cambridge, MA 02139 USA
[2] John Innes Inst, Dept Biol Chem, Norwich NR4 7UH, Norfolk, England
关键词
D O I
10.1021/ja036081r
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
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 °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. Copyright © 2003 American Chemical Society.
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页码:11150 / 11151
页数:2
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