Copper and topaquinone (TPQ) containing amine oxidases utilize O_2 for the metabolism of biogenic amines while concomitantly generating H_2O_2 for use by the cell. The mechanism of O_2 reduction has been the subject of long-standing debate due to the obscuring influence of a proton-coupled electron transfer between the tyrosine-derived TPQ and copper, a rapidly established equilibrium precluding assignment of the enzyme in its reactive form. Here, we show that substrate-reduced pea seedling amine oxidase (PSAO) exists predominantly in the Cu~1, TPQ semiquinone state. A new mechanistic proposal for O_2 reduction is advanced on the basis of thermodynamic considerations together with kinetic studies (at varying pH, temperature, and viscosity), the identification of steady-state intermediates, and the analysis of competitive oxygen kinetic isotope effects, ~(18)O KIEs, [k_(cat)/K_M(~(16, 16)O_2)]/[k_(can)/K_M~(16, 18)O_2)]. The ~(18)O KIE = 1.0136 ± 0.0013 at pH 7.2 is independent of temperature from 5℃ to 47℃and insignificantly changed to 1.0122 ± 0.0020 upon raising the pH to 9, thus indicating the absence of kinetic complexity. Using density functional methods, the effect is found to be precisely in the range expected for reversible O_2 binding to Cu~1 to afford a superoxide, [Cu~(II)(η~1-O_2)~(-1)]~+, intermediate. Electron transfer from the TPQ semiquinone follows in the first irreversible step to form a peroxide, Cu~(II)(η~1-O_2)~(II), intermediate driving the reduction of O_2. The similar ~(18)O KIEs reported for copper amine oxidases from other sources raise the possibility that all enzymes react by related inner-sphere mechanisms although additional experiments are needed to test this proposal.
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