Inner-Sphere Mechanism for Molecular Oxygen Reduction Catalyzed by Copper Amine Oxidases

Abstract

Copper and topaquinone (TPQ) containing amine oxidases utilize O2 for the metabolism of biogenic amines while concomitantly generating H2O2 for use by the cell. The mechanism of O2 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(I), TPQ semiquinone state. A new mechanistic proposal for O2 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, [kcat/KM((16,16)O2)]/[kcat/KM((16,18)O2)]. The (18)O KIE = 1.0136 +/- 0.0013 at pH 7.2 is independent of temperature from 5 degrees C to 47 degrees C 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 O2 binding to Cu(I) to afford a superoxide, [Cu(II)(eta(1)-O2)(-I)](+), intermediate. Electron transfer from the TPQ semiquinone follows in the first irreversible step to form a peroxide, Cu(II)(eta(1)-O2)(-II), intermediate driving the reduction of O2. 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.

Probing the Catalytic Mechanism of Copper Amine Oxidase from Arthrobacter globiformis with Halide Ions

“…The exclusive binding of halide ions to Cu(II) with TPQ amr in the off-copper conformation (which does not bind dioxygen) is challenging to reconcile with their activity as inhibitors of the oxidative half-reaction that are noncompetitive with respect to dioxygen. This issue can be resolved by supposing that dioxygen binds directly to Cu(I) with TPQ sq in the on-copper conformation during the oxidative half-reaction and undergoes 1 e Ϫ -reduction by Cu(I) through the inner sphere mechanism proposed for AGAO (37,38). Alternatively, halide ions may inhibit the oxidative halfreaction by binding to the O 2 -bound enzyme.…”

“…However, the mechanistic role of the TPQ sq ⅐ Cu(I) form in the subsequent O 2 reduction remains unclear and controversial (32)(33)(34)(35)(36)(37)(38)(39). Two reaction pathways for the O 2 reduction have been proposed, depending on the enzyme sources.…”

“…The necessary singlet to triplet spin transition is allowed by the coordination of O 2 to Cu(I). AGAO and P. savitum CAO are CAOs that have been suggested to follow this mechanism (37,38). The formation of the TPQ sq ⅐Cu(I) state at the beginning of the oxidative half-reaction is believed to be essential in this process.…”

Structural Snapshots from the Oxidative Half-reaction of a Copper Amine Oxidase

“…Therefore, it was suggested that the Co(II)-AGAO, pea seedling CAO, and ECAO enzymes use the less efficient mechanism of outersphere electron transfer for O 2 activation. However, the existence of an outer-sphere electron transfer mechanism in either copperor cobalt-containing CAOs has been recently challenged in pea seedling CAO and AGAO based on 18 O KIE studies and density functional theory (15,16), coupled with the smaller than expected 18 O KIE observed in HPAO-1 and bovine serum amine oxidase that are more in line with inner-sphere electron transfer (18,24). However, it should be noted that a larger 18 O KIE than observed in CAO is seen in the Cu(I) inner-sphere electron transfer activation of O 2 in galactose oxidase, indicating caution is warranted when linking 18 O KIE magnitude to the O 2 activation mechanism (52).…”

“…The information gained from these studies has led to a consensus mechanism for the amine oxidation to aldehyde (10). However, the mechanism of molecular oxygen activation remains the subject of controversy (11)(12)(13)(14)(15)(16). Kinetic isotope effects, steady-state kinetics, viscosogen, and stopped-flow experiments have shown that the reduction of molecular oxygen contributes 29% to the overall k cat of catalysis in Hansenula polymorpha amine oxidase 1 (HPAO-1) 3 (17,18).…”