Inhibition of amine oxidases by Cu(II) complexes and anions: Mechanistic implications

Abstract Copper-containing amine oxidases catalyze the oxidative deamination of primary amines by the following mechanism [1]: Our interests center on the role(s) of copper in reactions (1) and (2) and on the activation and utilization of O 2 by this enzymes. An important mechanistic question in this regard is whether then oxidation of E red proceeds via sequential one-electron steps or via a single, two-electron step. One-electron oxidation would generate O 2 − as an intermediate, whereas a two-electron oxidation [2] would not. Accordingly we investigated several Cu(II) complexes, previously shown to be superoxide dismutase active, as potential inhibitors of bovine plasma and pig kidney amine oxidase [3, 4]. Cu(II) complexes of 1, 10-phenanthroline (phen) and 2,2′-bipyridine (bipy) strongly inhibited both amine oxidases. Lysine, tyrosine, and salicylate complexes, as well as Cu 2+ (aq.), inhibited the plasma but not the kidney amine oxidase. No inhibition by the free ligands, by Cu(EDTA) 2− , or bovine erythrocyte SOD was observed. Cu(phen) 2 (NO 3 ) 2 , Cu 2+ (aq.), and Cu(salicylate) 2 displayed linear noncompetitive inhibition against amine substrate at saturating O 2 concentrations with either pig kidney or beef plasma amine oxidase. Since neither the free ligands nor Cu 2+ (aq.) inhibit the kidney enzyme at concentrations where Cu(phen) 2 (NO 3 ) 2 and Cl 2 are effective, the complexes. Two plausible inhibition mechanism are (1) dismutation of O 2 − , which is an intermediate in the reoxidation reaction; or (2) direct oxidation E red by the Cu(II) complexes. Steric requirements for the second should be mechanism should be less restrictive than for mechanism (1) as outer-sphere electron transfer is possible over substantial distances, but O 2 − dismutation is an inner-sphere process that requires the Cu(II) complex to penetrate to the O 2 reduction site. Thus if O 2 − is tightly bound within the dioxygen-reductions site, its accessibility to Cu(II) complexes of varying size and charge would be mechanically important. On this basis the reactivity pattern we have observed can be rationalized: only those complexes with hydrophobic ligands and a net positive charge inhibited pig kidney amine oxidase, but the active site in the plasma enzyme may be much more accessible to external reagents. N 3 − and SCN − are Cu(II)-specific inhibitors of several amine oxidases [1, 5–7]. Ligand-to-metal charge-transfer bands are evident at ∼365 nm (ϵ ∼3,000 M −1 cm −1 and ∼400 nm (ϵ ∼6,000 M −1 cm −1 when SCN − and N 3 − , respectively, are added to beef plasma or pig kidney amine oxidase. The intensities and energies establish that the anions are equitorially coordinated. Changes in the Cu(II) ligand field, as judged by absorption, circular dichroism and EPR spectroscopy, are consistent with a simple ligand substitution reaction. Preliminary NMR evidence indicates that H 2 O is the leaving group, as previously demonstrated for the pig plasma enzyme [6]. Depending on the amine concentration both the anions are linear noncompetitive or uncompetitive inhibitors of beef plasma amine oxidase with O 2 saturating; under the same conditions these anions were strictly linear uncompetitive inhibitors of the pig kidney enzyme. By determining in detail the inhibition mechanism of anions and the Cu(II) complexes, we should be able to learn much about the role(s) of copper in the amine oxidases.