Oxygen Kinetic Isotope Effects in Soluble Methane Monooxygenase

Abstract

Soluble methane monooxygenase (sMMO) contains a nonheme, carboxylate-bridged diiron site that activates dioxygen in the catalytic oxidation of hydrocarbon substrates. Oxygen kinetic isotope effects (KIEs) have been determined under steady-state conditions for the sMMO-catalyzed oxidation of CH 3 CN, a liquid substrate analog. Kinetic studies of the steady-state sMMO reaction revealed a competition between fully coupled oxygenase activity, which produced glycolonitrile (HOCH 2 CN) and uncoupled oxidase activity that led to water formation. The oxygen KIE was measured independently for both the oxygenase and oxidase reactions, and values of 1.0152 ؎ 0.0007 and 1.0167 ؎ 0.0010 were obtained, respectively. The isotope effects and separate dioxygen binding studies do not support irreversible formation of an enzyme-dioxygen Michaelis complex. Additional mechanistic implications are discussed in the context of previous data obtained from single turnover and steady-state kinetic studies.The soluble methane monooxygenase system (sMMO) 1 found in certain methanotrophic bacteria catalyzes the oxidation of methane to methanol according to Reaction 1 (1, 2).This reaction is the first essential step of a metabolic pathway that allows these bacteria to use methane as their sole source of energy and carbon. The soluble MMO system comprises three protein components: a 251 kDa hydroxylase (MMOH), a reductase (MMOR) that transfers electrons from NADH to MMOH, and a cofactorless regulatory protein (MMOB). Reductive activation of dioxygen and subsequent methane oxidation take place at a carboxylate-bridged diiron cluster in the active site of MMOH. (3,4,6,8). This observation suggests that Q, or a subsequent unobserved intermediate, is responsible for methane oxidation.Recent kinetic studies and theoretical calculations have suggested the presence of additional intermediates (6, 9, 10). For example, proton transfer steps have been observed in the formation and decay of H peroxo , a result consistent with a protonated peroxo intermediate in the mechanism (9). Other kinetic studies provide indirect evidence for one or more intermediates preceding the formation of H peroxo . These may include an enzyme-dioxygen Michaelis complex or a superoxo intermediate (6, 9 -11).Kinetic and equilibrium oxygen isotope effects have been determined recently for several proteins and enzymes that activate dioxygen (12)(13)(14)(15)(16)(17)(18). Such experiments directly probe reaction steps involved in the activation of dioxygen and can provide unique mechanistic information that may not be available by other means. In this study, oxygen-18 kinetic isotope effects determined for sMMO from M. capsulatus (Bath) together with steady-state kinetics and dioxygen binding studies yield further insights into dioxygen activation by this enzyme system. EXPERIMENTAL PROCEDURESGeneral Considerations-Growth of M. capsulatus (Bath) cells and subsequent purification of MMOH were carried out as described previously (19,20). Both MMOB and MMOR were obtained from recombi...

Mechanism of O2 Activation by Cytochrome P450cam Studied by Isotope Effects and Transient State Kinetics

“…When saturated with its redox partners, P450cam appears similar to most other O 2 -utilizing enzymes studied to date, including glucose oxidase (21)(22)(23), amine oxidase (24)(25)(26), tyrosine hydroxylase (27), and methane monooxygenase (28), in that reduction of O 2 by one electron contributes to the rate-limiting step in O 2 activation. It has been postulated that this serves to minimize accumulation of reactive oxygen species during catalysis (20).…”

“…These studies include reactions between O 2 and organic cofactors in glucose oxidase (21)(22)(23), amine oxidase (24)(25)(26), and tyrosine hydroxylase (27) or with a binuclear iron center in methane monooxygenase (28). In each of these cases, the 16 O/ 18 O kinetic isotope effect (KIE) on k cat /K m (O 2 ) was an important factor in determining the nature of the first committed step in O 2 activation.…”

Structures of Transition States in Metal‐Mediated O2‐Activation Reactions

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Enrichment of heavy oxygen ( 18 O) from its level at natural abundance is widely used to probe O 2 -dependent processes in the atmospheric and biological sciences.[ [4,[8][9][10][11] have been interpreted on the basis of equilibrium isotope effects (EIEs) and simple models that neglect variations in the transition-state structure. The present studies suggest that the proposed enzyme mechanisms may need to be reevaluated in light of the sensitivity of 18 O KIEs to changes in the height of the free-energy barrier for reactions involving the formation of a metal-O 2 bond.

Oxygenation

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Tracking a defined route for O 2 migration in a dioxygen-activating diiron enzyme

“…First, the dioxygen access or binding step has never been directly observed. Upon the reaction of reduced sMMOH with dioxygen in the presence of its regulatory protein, the first oxygenated species to appear does so at rates much slower than the decay rate of the reduced diiron centers, indicating that O 2 consumption is coupled with, or preceded by, slower step (s), possibly structural reorganization or formation of uncharacterized intervening species (19,20). In addition, a recombinant expression system to produce sMMOH variants in sufficient quantities for detailed kinetic studies has never been achieved.…”