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

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 nitro benzene to nitrophenol under both single turnover and catalytic condit ions. During catalytic turnover, hydroxylation occurs to afford 3-nitr ophenol (43%) and 4-nitrophenol (57%), whereas hydroxylation takes pla ce exclusively (>95%) to give 4-nitrophenol under single turnover cond itions in the absence of reductase. Protein B exerts a strong influenc e on single turnover reactions of nitrobenzene, with optimal rate cons tants and yields obtained by using 1.5-2 equiv of protein B per equiva lent of hydroxylase. The reaction of H-red and protein B with dioxygen in the absence of substrate was investigated by using stopped-flow an d freeze-quench methodology. In Mossbauer experiments, two distinct po pulations of diiron sites in H-red were detected which react with diox ygen on different time scales. Deconvolution of the time-dependent Mos sbauer spectra allowed the isomer shift (delta) and quadrupole splitti ng (Delta E(Q)) parameters as well as the kinetic constants to be extr acted for each species. For the faster reacting, physiologically relev ant component H-red(1), two kinetically competent intermediates were i dentified. 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 c onstant 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 forma tion and decay of the second intermediate, Q, which absorbs with lambd a(max) approximate to 350 and 420 nm and can also be followed by kinet ic 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(for m) 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 d etermined. Changes in dioxygen concentration and pH, as well as exchan ge of solvent accessible protons with D2O, did not significantly affec t the rate constants for either of these processes, the implications o f which for the kinetic mechanism are discussed. From the present and related evidence, structures for H-peroxo and Q are proposed.