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.

Figure 1

Oxidized forms of copper amine oxidase from pea seedling (a) and H. polymorpha (b).

Figure 2

The redox equilibrium in reduced PSAO (E_red) involving proton-coupled electron transfer.

Figure 3

(a) Redox titration of PSAO showing formation of TPQ_sq under anaerobic conditions.(b) The pH dependence of K_int (cf. Figure 2) corresponding to production of E(Cu^I, TPQ_sq) at 22 °C, μ = 0.1 M.

Figure 4

Profiles of (a) k_cat/K_M(O₂) and (b) k_cat at 22 °C (H₂O, red circles), (D₂O, blue squares). The ionic strength was maintained in all experiments (μ = 0.1 M).

Figure 5

Oxygen isotope fractionation during PSAO turnover at pH 7.2 and 22 °C, μ = 0.1 M.

Figure 6

Activation parameters for putrescine oxidation at pH 7.2 (μ = 0.1M): (a) the temperature dependence of (a) k_cat/K_M(O₂) and (b) k_cat. The k_cat values were determined by varying amine (open circles) or varying O₂ (closed circles). Error bars are ± 2σ.

Figure 7

Steady-state accumulation of the product Schiff-base at pH 7.2, μ = 0.5 M and 20°C.

Figure 8

The approach to steady-state in the reaction of PSAO (10 μM) with O₂ (1.0 mM) and putrescine (5 mM) at pH 7.2, μ = 0.1M and 24°C. (a) Overlay of spectra collected over 1 s. (b) The absorbance change at 462nm. (c) The initial and final calculated spectra (see text for details). (d) The changes in concentration corresponding to the initial and final spectra in (c).

Scheme 1

Kinetic mechanism for the oxidative half-reaction of PSAO.

Scheme 2

Thermodynamic cycle for assessing the driving-force for TPQ_red oxidation.