X-ray snapshots of quinone cofactor biogenesis in bacterial copper amine oxidase

Abstract

The quinone cofactor TPQ in copper amine oxidase is generated by posttranslational modification of an active site tyrosine residue. Using X-ray crystallography, we have probed the copper-dependent autooxidation process of TPQ in the enzyme from Arthrobacter globiformis. Apo enzyme crystals were anaerobically soaked with copper; the structure determined from this crystal provides a view of the initial state: the unmodified tyrosine coordinated to the bound copper. Exposure of the copper-bound crystals to oxygen led to the formation of freeze-trapped intermediates; structural analyses indicate that these intermediates contain dihydroxyphenylalanine quinone and trihydroxyphenylalanine. These are the first visualized intermediates during TPQ biogenesis in copper amine oxidase.

Copper Active Sites in Biology

“…In some AGAO structures, two conformations are observed for the residue His592, which is Nδ-coordinated to the copper. 1194,1195 This conformational flexibility, which is not observed in AOs from other sources, is proposed to be due to incomplete occupancy of the site by copper and likely is not significant.…”

Substrate activation for O 2 reactions by oxidized metal centers in biology

“…The net result of the O 2 attack is generally thought to be the formation of a peroxy-adduct bridging between the iron site and one of the hydroxylated carbons of the now quinone that requires a spin-forbidden two-electron transfer of the electron pair of the substrate to the triplet O 2 molecule (23). It is important to determine the mechanism of substrate activation for O 2 reaction in the intradiol dioxygenases both for this class of enzymes and for others where similar mechanisms have been invoked as, for example, in cofactor biogenesis in copper-containing amine oxidases (34,35).…”

Crystal Structure of Amine Oxidase from Bovine Serum

“…In BSAO, in particular, it could help to reveal the positions where molecular oxygen binds, both for the oxidation of TPQ during the catalytic cycle and the oxidation of Tyr470 in the TPQ biogenesis process. 18,32 An anomalous difference-Fourier map, calculated with phases obtained from the refined coordinates of the final model, shows 70 peaks larger than 4s. The first four maxima correspond to the position of the secondary cations, the fifth site corresponds to Cu(II) of the A subunit, the others to sulfur atoms of Met or Cys or to peaks that were interpreted as Xe atoms.…”

“…Each subunit contains a copper ion and a covalently bound redox cofactor, 2,4,5-trihydroxyphenylalanine quinone (topaquinone, TPQ), 16 formed within the active-site by oxidation of a single tyrosine residue, conserved in all CAOs. 17,18 The catalytic mechanism of CAOs has been thoroughly investigated over the last many years. On the basis of the results of kinetic and spectroscopic studies, [19][20][21][22][23][24][25] as well as structural and functional studies using site-directed mutant variants, [26][27][28] the catalytic mechanism is now quite well established.…”