Evidence That Dioxygen and Substrate Activation Are Tightly Coupled in Dopamine -Monooxygenase

Oxygen activation occurs at a wide variety of enzyme active sites. Mechanisms previously proposed for the copper monooxygenase, dopamine -monooxygenase (DM), involve the accumulation of an activated oxygen intermediate with the properties of a copper-peroxo or copper-oxo species before substrate activation. These are reminiscent of the mechanism of cytochrome P-450, where a heme iron stabilizes the activated O2 species. Herein, we report two experimental probes of the activated oxygen species in DM. First, we have synthesized the substrate analog, ,-difluorophenethylamine, and examined its capacity to induce reoxidation of the prereduced copper sites of DM upon mixing with O2 under rapid freeze-quench conditions. This experiment fails to give rise to an EPR-detectable copper species, in contrast to a substrate with a C–H active bond. This indicates either that the reoxidation of the enzyme-bound copper sites in the presence of O2 is tightly linked to C-H activation or that a diamagnetic species Cu(II)–O2 has been formed. In the context of the open and fully solvent-accessible active site for the homologous peptidylglycine--hydroxylating monooxygenase and by analogy to cytochrome P-450, the accumulation of a reduced and activated oxygen species in DM before C-H cleavage would be expected to give some uncoupling of oxygen and substrate consumption. We have, therefore, examined the degree to which O2 and substrate consumption are coupled in DM using both end point and initial rate experimental protocols. With substrates that differ by more than three orders of magnitude in rate, we fail to detect any uncoupling of O2 uptake from product formation. We conclude that there is no accumulation of an activated form of O2 before C-H abstraction in the DM and peptidylglycine--hydroxylating monooxygenase class of copper monooxygenases, presenting a mechanism in which a diamagnetic Cu(II)-superoxo complex, formed initially at very low levels, abstracts a hydrogen atom from substrate to generate Cu(II)-hydroperoxo and substrate-free radical as intermediates. Subsequent participation of the second copper site per subunit completes the reaction cycle, generating hydroxylated product and water.