Role of Copper Ion in Bacterial Copper Amine Oxidase:  Spectroscopic and Crystallographic Studies of Metal-Substituted Enzymes

Abstract

The role of the active site Cu(2+) of phenylethylamine oxidase from Arthrobacter globiformis (AGAO) has been studied by substitution with other divalent cations, where we were able to remove >99.5% of Cu(2+) from the active site. The enzymes reconstituted with Co(2+) and Ni(2+) (Co- and Ni-AGAO) exhibited 2.2 and 0.9% activities, respectively, of the original Cu(2+)-enzyme (Cu-AGAO), but their K(m) values for amine substrate and dioxygen were comparable. X-ray crystal structures of the Co- and Ni-AGAO were solved at 2.0-1.8 A resolution. These structures revealed changes in the metal coordination environment when compared to that of Cu-AGAO. However, the hydrogen-bonding network around the active site involving metal-coordinating and noncoordinating water molecules was preserved. Upon anaerobic mixing of the Cu-, Co-, and Ni-AGAO with amine substrate, the 480 nm absorption band characteristic of the oxidized form of the topaquinone cofactor (TPQ(ox)) disappeared rapidly (< 6 ms), yielding the aminoresorcinol form of the reduced cofactor (TPQ(amr)). In contrast to the substrate-reduced Cu-AGAO, the semiquinone radical (TPQ(sq)) was not detected in Co- and Ni-AGAO. Further, in the latter, TPQ(amr) reacted reversibly with the product aldehyde to form a species with a lambda(max) at around 350 nm that was assigned as the neutral form of the product Schiff base (TPQ(pim)). Introduction of dioxygen to the substrate-reduced Co- and Ni-AGAO resulted in the formation of a TPQ-related intermediate absorbing at around 360 nm, which was assigned to the neutral iminoquinone form of the 2e(-)-oxidized cofactor (TPQ(imq)) and which decayed concomitantly with the generation of TPQ(ox). The rate of TPQ(imq) formation and its subsequent decay in Co- and Ni-AGAO was slow when compared to those of the corresponding reactions in Cu-AGAO. The low catalytic activities of the metal-substituted enzymes are due to the impaired efficiencies of the oxidative half-reaction in the catalytic cycle of amine oxidation. On the basis of these results, we propose that the native Cu(2+) ion has essential roles such as catalyzing the electron transfer between TPQ(amr) and dioxygen, in part by providing a binding site for 1e(-)- and 2e(-)-reduced dioxygen species to be efficiently protonated and released and also preventing the back reaction between the product aldehyde and TPQ(amr).

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

“…6, B and C) still lies in the observation that Co(II)-substituted HPAO-1 has nearly identical k cat values to the native form (18). In contrast to HPAO-1, Co(II)-substituted AGAO, pea seedling CAO, and ECAO all have very low catalytic activities (15,(47)(48)(49). The catalytic rates are similar to wild-type copper-containing HPAO-1, HPAO-2, and bovine serum amine oxidase, which are also posited to employ an outer-sphere electron transfer mechanism from aminoquinol to activate O 2 (24,50,51).…”

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

“…Spectrophotometric Measurements-To achieve fully anaerobic conditions, the enzyme and substrate solutions were kept in a vacuum-type glove box (Iuchi, SGV-65V) filled with 99.999% (v/v) argon gas for at least 2 h, as described previously (18). The enzyme (final concentration, 100 M monomer) was anaerobically mixed with 1 mM 2-PEA in 50 mM HEPES, pH 6.8, in the presence or absence of various concentrations of sodium, potassium, or ammonium salts of halide ions.…”

“…As summarized in a recent review (3), x-ray crystal structures of CAOs have been determined for a number of enzymes from various sources, including bacteria (Escherichia coli (16) and Arthrobacter globiformis (AGAO) (17)(18)(19)), yeasts (Hansenula polymorpha (recently reclassified as Pichia angusta) (HPAO-1 and HPAO-2) (20,21) and Pichia pastoris (lysyl oxidase) (22)), a fungus (Aspergillus nidulans (23)), a plant (Pisum savitum (24)), a mammal (Bos taurus (25)), and Homo sapiens (diamine oxidase and vascular adhesion protein 1, which is identical to semicarbazide-sensitive amine oxidase (26 -28)). The active site structures of these enzymes, including the positions of TPQ and Cu(II), are highly conserved, suggesting that they have a common mechanism for single-turnover TPQ biogenesis and catalytic amine oxidation.…”

Cobalt substitution supports an inner-sphere electron transfer mechanism for oxygen reduction in pea seedling amine oxidase

“…One method (used for HPAO) uses an amine substrate to reduce the metal [8]. The other (used for BSAO and AGAO) uses dithionite to reduce the copper [15,17]. Both methods were successful in removing the copper from PSAO; however, the dithionite method removed more copper (96%, see Table 1) than the method using substrate (85%, results not shown).…”

“…Copperdepleted PSAO (CuDepPSAO) was prepared following a modification of the method used for AGAO [15]. PSAO (2.5 mL, 1.2 mg mL -1 ) was made anaerobic and introduced into a Slide-a-Lyzer in an anaerobic chamber.…”