Mechanistic studies of the reaction of reduced methane monooxygenase hydroxylase with dioxygen and substrates
- Massachusetts Inst. of Tech., Cambridge, MA (United States). Dept. of Chemistry
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{sub peroxo} is the first intermediate observed in the reaction between the chemically reduced hydroxylase (H{sub 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 {lambda}{sub max} {approx} 420 nm ({epsilon} = 4,000 M{sup {minus}1} cm{sup {minus}1}) and {lambda}{sub max} = 725 nm ({epsilon} = 1,800 M{sup {minus}1} cm{sup {minus}1}). The temperature dependences of the rate constants for formation and decay of H{sub 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 {lambda}{sub 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{sub 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{sub peroxo} formation, 1--2 s{sup {minus}1}, is slower than that determined previously by Moessbauer and optical spectroscopy, {approximately}20--25 s{sup {minus}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{sub peroxo} can oxidize olefins but not acetylene or saturated hydrocarbons.
- OSTI ID:
- 684319
- Journal Information:
- Journal of the American Chemical Society, Vol. 121, Issue 16; Other Information: PBD: 28 Apr 1999
- Country of Publication:
- United States
- Language:
- English
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