KINETIC AND SPECTROSCOPIC CHARACTERIZATION OF INTERMEDIATES AND COMPONENT INTERACTIONS IN REACTIONS OF METHANE MONOOXYGENASE FROM METHYLOCOCCUS CAPSULATUS (BATH)

摘要

We describe mechanistic studies of the soluble methane monooxygenase (sMMO) enzyme system from Methylococcus capsulatus (Bath). Interactions among the three sMMO components, the hydroxylase (H), reductase (R), and protein B (B), were investigated by monitoring conversion of nitrobenzene to nitrophenol under both single turnover and catalytic conditions. During catalytic turnover, hydroxylation occurs to afford 3-nitrophenol (43%) and 4-nitrophenol (57%), whereas hydroxylation takes place exclusively (>95%) to give 4-nitrophenol under single turnover conditions in the absence of reductase. Protein B exerts a strong influence on single turnover reactions of nitrobenzene, with optimal rate constants and yields obtained by using 1.5-2 equiv of protein B per equivalent of hydroxylase. The reaction of H-red and protein B with dioxygen in the absence of substrate was investigated by using stopped-flow and freeze-quench methodology. In Mossbauer experiments, two distinct populations of diiron sites in H-red were detected which react with dioxygen on different time scales. Deconvolution of the time-dependent Mossbauer spectra allowed the isomer shift (delta) and quadrupole splitting (Delta E(Q)) parameters as well as the kinetic constants to be extracted for each species. For the faster reacting, physiologically relevant component H-red(1), two kinetically competent intermediates were identified. The first intermediate, H-peroxo (delta = 0.66 mm/s; Delta E(Q) = 1.51 mm/s), a diiron(III) peroxide species, forms with a rate constant of approximate to 25 s(-1) and decays with a rate constant of approximate to 0.45 s(-1) at 4 degrees C. Rate constants for the formation and decay of the second intermediate, Q, which absorbs with lambda(max) approximate to 350 and 420 nm and can also be followed by kinetic freeze-quench Mossbauer spectroscopy (delta = 0.21 mm/s; Delta E(Q) = 0.68 mm/s and Delta = 0.14 mm/s; Delta E(Q) = 0.55 mm/s), are k(form) approximate to 0.45 s(-1) and k(decay) approximate to 0.05 s(-1) at 4 degrees C. The temperature dependence of these kinetic values was determined. Changes in dioxygen concentration and pH, as well as exchange of solvent accessible protons with D2O, did not significantly affect the rate constants for either of these processes, the implications of which for the kinetic mechanism are discussed. From the present and related evidence, structures for H-peroxo and Q are proposed. [References: 55]