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J. Biochem, 1988, Vol. 104, No. 5 727-733
© 1988 Japanese Biochemical Society

research-article

Complex Formation between Anionic Semiquinoid Form of a Flavoenzyme D-Amino Acid Oxidase and Ligands. Stabilizing Mechanism of Anionic Semiquinoid Flavoenzyme1

Yasuzo Nishina*, Hiromasa Tojo**, Retsu Miura***,2, Yoshihiro Miyake*** and Kiyoshi Shiga*

*Department of Physiology, Kumamoto University Medical School Kumamoto, Kumamoto 860
**Department of Biochemistry, Osaka University Medical School Kita-ku, Osaka, Osaka 530
***Department of Biochemistry, National Cardiovascular Center Research Institute Suita, Osaka 565

Picolinate binds to the anionic semiquinoid form of D-amino acid oxidase (DAO), 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 623.8 nm of the anionic semiquinoid DAO complex formed in the presence of pyruvate and methylamine indicate that the complex consists of the anionic semiquinoid DAO and N-methyl-{alpha}-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 fiavin. 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 fiavin C(4a)-N(5) locus and the locus containing 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.

1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

2 Present address: Laboratory of Chemistry, Kansai Medical Univer sity, Hirakata, Osaka 573.


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