The interactions of cyanide with two copper-containing amine oxidases (CuAOs) from pea seedlings (PSAO) and the soil bacterium Arthrobacter globiformis (AGAO) have been investigated by spectroscopic and kinetic techniques. Previously, we rationalized the effects of azide and cyanide for several CuAOs in terms of copper coordination by these exogenous ligands and their effects on the internal redox equilibrium TPQ(amr)-Cu(II) right harpoon over left harpoon TPQ(sq)-Cu(I). The mechanism of cyanide inhibition was proposed to occur through complexation to Cu(I), thereby directly competing with O(2) for reoxidation of TPQ. Although cyanide readily and reversibly reacts with quinones, no direct spectroscopic evidence for cyanohydrin derivatization of TPQ has been previously documented for CuAOs. This work describes the first direct spectroscopic evidence, using both model and enzyme systems, for cyanohydrin derivatization of TPQ. K(d) values for Cu(II)-CN(-) and Cu(I)-CN(-), as well as the K(i) for cyanide inhibition versus substrate amine, are reported for PSAO and AGAO. In spite of cyanohydrin derivatization of the TPQ cofactor in these enzymes, the uncompetitive inhibition of amine oxidation is determined to arise almost exclusively through CN(-) complexation of Cu(I).

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The consensus mechanism for biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor in copper amine oxidases involves a key water addition to the dopaquinone intermediate. Although hydration of o-quinones seems straightforward and was implicated previously in aqueous autoxidation of catechols to give ultimately hydroxyquinones, a recent study (Mandal, S.; Lee, Y.; Purdy, M. M.; Sayre, L. M. J. Am. Chem. Soc. 2000, 122, 3574-3584) showed that the observed hydroxyquinones arise not from hydration, but from addition to the o-quinones of H(2)O(2) generated during autoxidation of the catechols. In the enzyme case, hydration of dopaquinone is proposed to be mediated by the active site Cu(II). To establish precedent for this mechanism, we engineered a catechol tethered to a Cu(II)-coordinating unit, such that the corresponding o-quinone could be generated in situ by oxidation with periodate (to avoid generation of H(2)O(2)). Thus, coordination of 4-((2-(bis(2-pyridylmethyl)amino)ethylamino)methyl)-1,2-benzenediol (1) to Cu(II) and subsequent addition of periodate resulted in rapid formation of the TPQ-like corresponding hydroxyquinone. Hydroxyquinone formation was seen also using Zn(II) and Ni(II), but not in the absence of M(II). Under the same conditions, periodate oxidation of the simple catechol 4-tert-butylcatechol does not give hydroxyquinone in the presence or absence of Cu(II). M(II)OH(2) pK(a) data for the Cu(II), Zn(II), and Ni(II) complexes with the pendant tetradentate ligand in the masked (dimethyl ether) catechol form, and kinetic pH-rate profiles of the metal-dependent hydroxyquinone formation from periodate oxidation of catechol 1, suggested a rate-limiting addition step of the ligand-coordinated M(II)OH to the o-quinone intermediate. This study represents the first chemical demonstration of a true o-quinone hydration, which occurs in cofactor biogenesis in copper amine oxidases.

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Although copper(II)-mediated oxidation of phenols results in oxidative coupling rather than in oxygenation, it was recently reported that naturally occurring 5-alkylresorcinols undergo oxygenation in the presence of copper(II). To explore the generality of this reaction, the copper(II)-mediated autoxidation of 4-tert-butylresorcinol and 4,6-di-tert-butylresorcinol was investigated and was found to result in direct oxygenation at open activated positions and, at the tert-butyl-substituted positions, in oxygenation with competing loss of (as isobutylene) and 1,2-rearrangement of the tert-butyl group. 5-tert-Butyl-2-hydroxy-1,4-benzoquinone is the major product from both starting materials, and the final product mixture reflects, in part, coupling of metastable initially formed electrophilic and nucleophilic side products. Mechanisms that are consistent with the observed products and control reactions are proposed. The key step appears to be equilibration of a copper(II)-resorcinolate with a charge-transfer radical form that reacts regioselectively with O(2) as prescribed by resonance.