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 lambda(max) approximate to 420 nm (epsilon = 4600 M-1 cm(-1)) and lambda(max) = 725 nm (epsilon = 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 lambda(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 i 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 degrees C for Hperoxo formation, 1-2 s(-1), is slower than that determined previously by Mossbauer and optical spectroscopy, similar to 20-25 s(-1) (Liu, K. E.; et al. J. Am. Chem. Sec. 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.