Intramolecular electron transfer rate between active-site copper and TPQ in Arthrobacter globiformis amine oxidase.

Copper amine oxidases catalyze the oxidative deamination of primary amines operating through a ping-pong bi bi mechanism, divided into reductive and oxidative half-reactions. Considerable debate still exists regarding the role of copper in the oxidative half-reaction, where O2 is reduced to H2O2. Substrate-reduced amine oxidases display an equilibrium between a Cu(II) aminoquinol and a Cu(I) semiquinone, with the magnitude of the equilibrium constant being dependent upon the enzyme source. The initial electron transfer to dioxygen has been proposed to occur from either the reduced Cu(I) center or the reduced aminoquinol cofactor. In order for Cu(I) to be involved, it must be shown that the rate of electron transfer (kET) between the aminoquinol and Cu(II) is sufficiently rapid to place the Cu(I) semiquinone moiety on the mechanistic pathway. To further explore this issue, we measured the intramolecular electron transfer rate for the Cu(II) aminoquinol left arrow over right arrow Cu(I) semiquinone equilibrium in Arthrobacter globiformis amine oxidase (AGAO) by temperature-jump relaxation techniques. The results presented herein establish that kET is greater than the rate of catalysis (kcat) for the preferred amine substrate beta-phenylethylamine at three pH values, thereby permitting the Cu(I) semiquinone to be a viable catalytic intermediate during enzymatic reoxidation in this enzyme. The data show that kET is approximately equivalent at pH 6.2 and 7.2, being 2.5 times kcat for these pH values. At pH 8.2, however, kET decreases, becoming comparable to kcat. Potential reasons for the decreased kET at basic pH are presented. The implications of these results in light of a previously published study measuring reoxidation rates of substrate-reduced AGAO are also addressed.