| Monooxygenases catalyze a ubiquitous reaction in nature; the reductive cleavage of dioxygen, linked to the insertion of an oxygen atom into a C-H bond and concomitant release of water. The non-coupled binuclear copper proteins dopamine beta-monooxygenase (DbetaM) and peptidylglycine alpha-hydroxylating monooxygenase (PHM) comprise a unique class of monooxygenase. The two Cu active sites are distant, and activation of O2 occurs at a single Cu center to generate a reactive Cu2+/O2 species for H-atom abstraction from the C-H bond of substrate. Previously proposed mechanisms involve the accumulation of activated-dioxygen intermediates with the properties of a copper-peroxo or -hydro-peroxo. Herein we seek to identify kinetically the activated copper-dioxygen species responsible for hydrogen atom abstraction using a combination of site directed mutagenesis, steady state kinetics and kinetic isotope effects with natural substrates and substrate analogs.;The substrate analog beta,beta-difluorophenethylamine, fails to induce reoxidation of the prereduced copper sites of DbetaM upon mixing with O 2 under rapid freeze-quench conditions, in contrast to a substrate with a C-H active bond. 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 DbetaM, and propose a mechanism in which a diamagnetic Cu2+-superoxo complex, formed initially at very low levels, abstracts a hydrogen atom from substrate to generate Cu 2+-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.;Taking full advantage of the high level expression system of PHM, an essential histidine ligand to the electron transfer copper (CuA) was mutated to an alanine, and found to retain copper binding and hydroxylase activity. An extensive kinetic and deuterium isotope effect study found this mutant to maintain full coupling of O2 consumed to product formed despite a three order-of-magnitude decrease in kcat and a ∼300-fold decrease in kcat/ Km(O2). Unexpectedly, electron transfer is not rate limiting in H172A. Rather, the increased kinetic isotope effect (KIE) on kcat of 3.27 +/- 0.39, suggests that C-H bond cleavage has become more rate limiting, implicating a role for His 172 that is more than as a simple Cu ligand. (Abstract shortened by UMI.). |
