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).

Cu-containing nitrite reductase is a homotrimer in which a ca. 36 kDa monomer contains each of type 1 Cu (two His, Cys, and Met ligands) and type 2 Cu (three His and solvent ligands). Type 1 Cu receives one electron from an electron donor and transfers it to the reaction center, type 2 Cu. The distance between the two Cu atoms bound by the Cys-His sequence segment is 12.6 A. The intramolecular electron transfer from type 1 Cu to type 2 Cu occurs probably through this segment. The noncoordinated Asp and His residues around type 2 Cu play important roles in both the electron-transfer and the catalytic processes.

A heterologous expression system of the blue copper-containing nitrite reductase from Alcaligenes xylosoxidans GIFU1051 (AxgNIR) was constructed, and the purified recombinant enzyme was characterized. All the characteristic spectroscopic properties and enzyme activity of native AxgNIR were retained in the copper-reconstituted recombinant protein expressed in Escherichia coli, indicating the correct coordination of two types of Cu (type 1 and 2) in the recombinant enzyme. Moreover, two conserved noncoordinate residues, Asp98 and His255, located near the type 2 Cu site were replaced to elucidate the catalytic residue(s) of NIR. The Asp98 residue hydrogen-bonded to the water molecule ligating the type 2 Cu was changed to Ala, Asn, or Glu, and the His255 residue hydrogen-bonded to Asp98 through the water molecule was replaced with Ala, Lys, or Arg. The catalytic rate constants of all mutants were decreased to 0.4-2% of those of the recombinant enzyme, and the apparent K(m) values for nitrite were greatly increased in the Asp98 mutants. All the steady-state kinetic data of the mutants clearly demonstrate that both Asp98 and His255 are involved not only in the catalytic reaction but also in the substrate anchoring.