Peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) catalyzes the stereospecific hydroxylation of a glycyl alpha-carbon in a reaction that requires O-2 and ascorbate. Subsequent dealkylation of the alpha-hydroxyglycine by another enzyme, peptidylamidoglycolate lyase (PAL. EC 4.3.2.5), yields a bioactive amide and glyoxylate. PHM is a noncoupled, type II dicopper monooxygenase which activates 02 at only a single copper atom, Cu-M. In this study, the PHM mechanism was probed using a non-natural substrate, benzaldehyde imino-oxy acetic acid (BIAA). PHM catalyzes the O-oxidative dealkylation of BIAA to benzaldoxime and glyoxylate with no involvement of PAL. The minimal kinetic mechanism for BIAA was shown to be steady-state ordered using primary deuterium kinetic isotope effects. The D(V/K)(APPARENT), BIAA decreased from 14.7 +/- 1.0 as [O-2] -> 0 to 1.0 +/- 0.2 as [O-2] -> infinity suggesting the dissociation rate constant from the PHM.BIAA complex decreases as [O-2] increases; thereby, reducing the steady-state concentration of [PHM](free). BIAA was further used to differentiate between potential oxidative Cu/O species using a QM/ MM reaction coordinate simulation to determine which species could yield product O-dealkylation that matched our experimental data. The results of this study provided compelling evidence for the presence of a covalently linked Cu-II-alkoxide intermediate with a quartet spin state responsible BIAA oxidation.