Oxygen-18 kinetic isotope effects in the dopamine beta-monooxygenase reaction: evidence for a new chemical mechanism in non-heme metallomonooxygenases.

Previous studies of dopamine beta-monooxygenase (D beta M) have implicated the formation of a substrate-derived benzylic radical via a hydrogen atom abstraction mechanism [Miller & Klinman (1985) Biochemistry 24, 2114]. We now address the nature of the oxygen species catalyzing C-H bond cleavage through the measurement of oxygen-18 isotope effects as a function of substrate structure. Using deuterium isotope effects, together with experimental O-18 isotope effects with protonated and deuterated substrates, it has been possible to calculate intrinsic O-18 isotope effects. Since the D beta M mechanism includes many steps which may involve changes in bond order at dioxygen, e.g., the reversible binding of O2 to the active-site copper and its reductive activation to a copper-hydroperoxide species, the intrinsic O-18 isotope effect is expected to be the product of two terms: (1) an overall equilibrium O-18 isotope effect on steps leading from O2 binding to the formation of the intermediate which catalyzes C-H bond cleavage and (2) a kinetic O-18 isotope effect on the C-H bond cleavage step. Thus, the magnitude of a single O-18 isotope effect measurement cannot reveal the nature of the bonding at oxygen during substrate activation. In the present study we have measured the change in O-18 isotope effect as a function of substrate structure and reactivity, finding values of 18(V/K) which decrease from 1.0281 +/- 0.001 to 1.0216 +/- 0.0003 as the rate of the C-H bond cleavage step decreases from 680 to 2 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)