Complex Formation between Anionic Semiquinoid Form of a Flavoenzyme D-Amino Acid Oxidase and Ligands. Stabilizing Mechanism of Anionic Semiquinoid Flavoenzyme

Abstract

Picolinate binds to the anionic semiquinoid form of D-amino acid oxidase (IMO), and the complex formed has a broad absorption band in the long-wavelength region extending beyond 800 nm, which is reminiscent of a charge transfer interaction. The binding has a stoichiometry of 1:1 with respect to the enzyme. The dissociation constant at 25°C was 30μM at pH 7.0. The pH dependence (pH 7.0-8.3) of the dissociation constant indicates that one proton is associated with the complex formation, and suggests that picolinate able to bind to the anionic semiquinoid enzyme is in the cationic form protonated at the nitrogen atom. By adding dithionite to the oxidized DAO solution containing pyruvate and various amines, a similar anionic semiquinoid DAO complex having a broad long-wavelength absorption band, appeared. Resonance Raman spectra with excitation at 623.8nm of the anionic semiquinoid DAO complex formed in the presence of pyruvate and methylamine indicate that the complex consists of the anionic semiquinoid DAC, and N-methyl-α-iminopropionate produced from pyruvate and methylamine, and that the imino group must be protonated. This supports the proposal that the presence of a positively charged group in the vicinity of flavin is required for the stabilization of the anionic semiquinoid flavin. The results also suggest that the broad absorption band is derived from the charge transfer interaction between the anionic semiquinoid flavin and the imino acid, in which the flavin C (4a)-N (5) locus and the locus containing H-N+-=C-COO^- of the amino acid are important for the interaction. The charge-transfer complexes of anionic semiquinoid DAO hardly react with a reducing agent (dithionite or photoreduction) or an oxidizing agent (oxygen), different from free anionic semiquinoid DAO. These phenomena can be explained by the complexation (charge-transfer complex) with ligands over the flavin C (4a)-N(5) locus.