Functional analysis of the copper-dependent quercetin 2,3-dioxygenase. 2. X-ray absorption studies of native enzyme and anaerobic complexes with the substrates quercetin and myricetin.

Quercetin 2,3-dioxygenase (2,3QD) is a mononuclear copper-dependent dioxygenase which catalyzes the cleavage of the heterocyclic ring of the flavonol quercetin (5,7,3',4'-tetrahydroxy flavonol) to produce 2-protocatechuoyl-phloroglucinol carboxylic acid and carbon monoxide. In this study, X-ray absorption spectroscopy has been used to characterize the local structural environment of the Cu(2+) center of Aspergillus japonicus 2,3QD. Analysis of the EXAFS region of native 2,3QD at functionally relevant pH (pH 6.0) indicates an active site equally well-described by either four or five ligands (3N(His) + 1-2O) at an average distance of 2.00 A. Bond valence sum analysis confirms that the best model is somewhere between the two. When, however, 2,3QD is anaerobically complexed with its natural substrate quercetin, the copper environment undergoes a transition to a five-coordinated cage, which is also best modeled by a single shell of N/O scatterers at the average distance of 2.00 A. This coordination is independently confirmed by the anaerobic complex with myricetin (5'-hydroxy quercetin). XANES analysis confirms that substrate binding does not reduce the Cu(2+) ion. The present study gives the first direct insights into the coordination chemistry of the enzyme complexed with its substrates. It suggests that activation for O(2) attack is achieved by monodentate substrate complexation to the copper ion through the 3-hydroxyl group. In addition, monodentate carboxylate ligation by the Glu73 side chain is likely to play a role in the fine-tuning of the equilibrium leading to the formation of the activated E.S complex.