Mechanistic Studies of the Reaction of Reduced Methane Monooxygenase Hydroxylase with Dioxygen and Substrates

Abstract

Soluble methane monooxygenase (sMMO) catalyzes the oxidation of methane to methanol. Single-turnover reactions of sMMO from Methylococcus capsulatus (Bath) were studied by stopped-flow optical spectroscopy to examine further the activated dioxygen intermediates and their reactions with hydrocarbon substrates. A diiron(III) peroxo species designated H peroxo is the first intermediate observed in the reaction between the chemically reduced hydroxylase (H red ) and dioxygen. The optical spectrum of this species determined by diode array detection is presented for the first time and exhibits visible absorption bands with λ max ≈ 420 nm (ε = 4000 M -1 cm -1 ) and λ max = 725 nm (ε = 1800 M -1 cm -1 ). The temperature dependences of the rate constants for formation and decay of H peroxo and for the subsequent intermediate, Q, were examined in the absence and in the presence of hydrocarbon substrates, and activation parameters for these reactions were determined. For single-turnover reaction kinetics monitored at 420 nm, the λ max for Q, a nonlinear Eyring plot was obtained when acetylene or methane was present in sufficiently high concentration. This behavior reflects a two-step mechanism, Q formation followed by Q decay, in which the rate-determining step changes depending on the temperature. The rate of H peroxo formation does not depend on dioxygen concentration, indicating that an effectively irreversible step involving dioxygen precedes formation of the diiron(III) peroxo species. The rate constant observed at 4 °C for H peroxo formation, 1−2 s -1, is slower than that determined previously by Mössbauer and optical spectroscopy, ∼20−25 s -1 (Liu, K. E.; et al. J. Am. Chem. Soc. 1995, 117, 4997−4998; 10174−10185). Possible explanations for this discrepancy include the existence of two distinct peroxo species. Intermediate Q exhibits photosensitivity when monitored by diode array methodology, a property that may arise from enhanced reactivity of a transient charge-transfer species. The photoreaction can be avoided by using a monochromator to obtain kinetics data at single wavelengths. The reactions of substrates with intermediate species were studied by single- and double-mixing stopped-flow spectroscopy. The Q decay rate exhibits an approximate first-order dependence on substrate concentration for a wide range of hydrocarbons, the relative reactivity varying according to the sequence acetylene > ethylene > ethane > methane > propylene > propane. In addition, the data indicate that H peroxo can oxidize olefins but not acetylene or saturated hydrocarbons.