Characterization of the Preprocessed Copper Site Equilibrium in Amine Oxidase and Assignment of the Reactive Copper...

Copper-dependent amine oxidases produce their redox active cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), via the Cu^II-catalyzed oxygenation of an active site tyrosine. This study addresses possible mechanisms for this biogenesis process by presenting the geometric and electronic structure characterization of the Cu^II-bound, prebiogenesis (preprocessed) active site of the enzyme Arthrobacter globiformis amine oxidase (AGAO). Cu^II-loading into the preprocessed AGAO active site is slow ( k_obs = 0.13 h^-1), and is preceded by Cu^II binding in a separate kinetically favored site that is distinct from the active site. Preprocessed active site Cu^II is in a thermal equilibrium between two species, an entropically favored form with tyrosine protonated and unbound from the Cu^II site, and an enthalpically favored form with tyrosine bound deprotonated to the Cu^II active site. It is shown that the Cu^II-tyrosinate bound form is directly active in biogenesis. The electronic structure determined for the reactive form of the preprocessed Cu^II active site is inconsistent with a biogenesis pathway that proceeds through a Cu^I-tyrosyl radical intermediate, but consistent with a pathway that overcomes the spin forbidden reaction of ³O₂ with the bound singlet substrate via a three-electron concerted charge-transfer mechanism.