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

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Abstract
The role of the active site Cu2+ 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 Cu2+ from the active site. The enzymes reconstituted with Co2+ and Ni2+ (Co- and Ni-AGAO) exhibited 2.2 and 0.9% activities, respectively, of the original Cu2+-enzyme (Cu-AGAO), but their Km 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 Å 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 (TPQox) disappeared rapidly (< 6 ms), yielding the aminoresorcinol form of the reduced cofactor (TPQamr). In contrast to the substrate-reduced Cu-AGAO, the semiquinone radical (TPQsq) was not detected in Co- and Ni-AGAO. Further, in the latter, TPQamr reacted reversibly with the product aldehyde to form a species with a λmax at around 350 nm that was assigned as the neutral form of the product Schiff base (TPQpim). 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 (TPQimq) and which decayed concomitantly with the generation of TPQox. The rate of TPQimq 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 Cu2+ ion has essential roles such as catalyzing the electron transfer between TPQamr 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 TPQamr.