Oxygen Activation Switch in the Copper Amine Oxidase of Escherichia coli

Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu²⁺ in the active site, two peripheral calcium sites, ∼32 Å from the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca²⁺ (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Ca²⁺ (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Q mutation at the surface site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (∼60% WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity (∼4.6% WTECAO activity). To understand how these long-range mutations have major effects on TPQ biogenesis and catalysis, we employed ultraviolet-visible spectroscopy, steady-state kinetics, inhibition assays, and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu²⁺-substrate reduced TPQ (TPQ_AMQ) Cu⁺-TPQ semiquinone (TPQ_SQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu²⁺-TPQ_AMQ or destabilizes Cu⁺-TPQ_SQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu²⁺-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and while a Cu⁺-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.