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. CD
3NO
2 was then introduced in the second phase of the experiment, which was carried out in D
2O at 25
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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 NO
2 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.