Mechanism of Topa Quinone Biogenesis in Copper Amine Oxidase Studied by Site-Directed Mutagenesis and X-Ray Crystallography

Abstract

The copper-dependent, self-catalytic mechanism of the biogenesis of the topa quinone (TPQ) cofactor has been studied by site-specific mutagenesis of the three His residues (His431, His433, and His592) involved in binding of the Cu atom in the recombinant phenylethylamine oxidase from Arthrobacter globiformis. All three mutants, H431A, H433A, and H592A, mostly lacked the ability to form TPQ upon aerobic incubation with Cu2+ ion. However, free imidazole added exogenously to the H592A mutant rescued stable binding of the Cu2+ ion and TPQ formation, both of which were also confirmed by X-ray crystallography. In contrast, a tyrosinate-to-Cu2+ ion charge-transfer complex was formed transiently in the H433A mutant. These results demonstrate the functional non-equivalence of the three Cu-binding His residues and their importance for TPQ biogenesis.