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, similar to 32 angstrom from the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Cu2+ (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Cu2+ (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Qmutation 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 (similar to 60%WTECAO TPQ), I342F/E573Qhas almost no amine oxidase activity (similar to 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)) CutTPQsemiquinone (TPQ(SQ)) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu2+-TPQ(SQ) 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.
- INTRAMOLECULAR ELECTRON-TRANSFER
- OXIDATIVE HALF-REACTION
- OXIDASE/VASCULAR ADHESION PROTEIN-1
- QUINONE COFACTOR BIOGENESIS
- ACTIVE-SITE COPPER
- CATALYTIC MECHANISM
- TOPA QUINONE