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 O-3(2) with the bound singlet substrate via a three-electron concerted charge-transfer mechanism.