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

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