Oxygen Activation Switch in the Copper Amine Oxidase of Escherichia coli

Abstract

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 (TPQAMQ) Cu+-TPQ semiquinone (TPQSQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu2+-TPQAMQ or destabilizes Cu+-TPQSQ. 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.

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

“…Preprocessed AGAO contains three types of Cu II species, one in the kinetic binding site distinct from the active site and two thermodynamically favored active site species. Given that the kinetic binding site is distinct from the active site, it is possible that it corresponds to the putative calcium binding site recently elucidated in the homologue ECAO . The two thermodynamically favored Cu II site species are in a temperature dependent equilibrium.…”

“…Given that the kinetic binding site is distinct from the active site, it is possible that it corresponds to the putative calcium binding site recently elucidated in the homologue ECAO. 37 The two thermodynamically favored Cu II site species are in a temperature dependent equilibrium. The minor form of this equilibrium is enthalpically favored and entropically disfavored relative to the major form, so that at room temperature it is present at only 5% relative to the major form.…”

Copper‐Catalyzed Monooxygenation of Phenols: Evidence for a Mononuclear Reaction Mechanism

“…Addition of substrate and base to this species leads to liberation of 3,5‐di‐ tert ‐butylsemiquinone (DTBSQ) and formation of a phenolate‐copper(II) complex (Scheme 4b), starting the actual catalytic cycle. Following the mechanism of TPQ biosynthesis, [32, 35, 45] the phenolate ligand in intermediate b converts to a phenoxyl radical, reducing Cu II to Cu I . The Cu I phenoxyl complex (Scheme 4c) reacts with O 2 to give d , a bridging peroxo intermediate, which is subsequently transformed to the o ‐quinone complex (Scheme 4e) by O−O cleavage.…”

Kupfer‐katalysierte Monooxygenierung von Phenolen: Evidenz für einen mononuklearen Reaktionsmechanismus

“…Während die vorgelagerte Reaktionssequenz sowohl über einen mono‐ als auch einen dinuklearen Mechanismus verlaufen kann (siehe unten), schließt die Tatsache, dass der katalytische Zyklus auf Cu II basiert, einen dinuklearen Mechanismus aus (die Bildung eines μ‐η 2 :η 2 ‐ Peroxo‐Intermediats erfolgt aus zwei Cu I ‐Zentren und O 2 ). Stattdessen kann angenommen werden, dass er dem anderen bekannten Szenario für die Monooxygenierung von Phenolen in der Natur, der Biosynthese von TPQ in AO, folgt, das mononuklear ist (siehe oben) [30–35, 45, 46] . Entsprechend dieser Überlegung gehen wir davon aus, dass das Cu I ‐Salz mit O 2 und einem Substratmolekül während der Induktionsperiode zu einem Cu II ‐Semichinon‐Komplex reagiert (Schema 4, a ).…”

“…Stattdessen kann angenommen werden, dass er dem anderen bekannten Szenario für die Monooxygenierung von Phenolen in der Natur, der Biosynthese von TPQ in AO, folgt, das mononuklear ist (siehe oben). [30-35, 45, 46] Entsprechend dieser Überlegung gehen wir [32,35,45] [9,25] vorhanden ist, aber ohne Überschuss, was zu einem Austausch des gebundenen Substrats führen könnte.…”