Stopped-Flow Fourier Transform Infrared Spectroscopy of Nitromethane Oxidation by the Diiron(IV) Intermediate of Methane Monooxygenase

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Abstract
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.