title: 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).
authors.affiliation: Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Sei'ichiro
authors.initials: S
authors.lastname: Kishishita
authors.forename: Toshihide
authors.initials: T
authors.lastname: Okajima
authors.forename: Misa
authors.initials: M
authors.lastname: Kim
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Shun
authors.initials: S
authors.lastname: Hirota
authors.forename: Shinnichiro
authors.initials: S
authors.lastname: Suzuki
authors.forename: Shun'ichi
authors.initials: S
authors.lastname: Kuroda
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Tanizawa
authors.forename: Minae
authors.initials: M
authors.lastname: Mure
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry* / Q000378:metabolism; D001173:Arthrobacter / Q000201:enzymology; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D007700:Kinetics; D008958:Models, Molecular; D009532:Nickel / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
chemical_list: D011994:Recombinant Proteins; D003035:Cobalt; D003300:Copper; D009532:Nickel; C086534:phenylethylamine oxidase; D006631:Amine Oxidase (Copper-Containing)