Mechanism of enzyme action. III. Crystallization of the semiquinoid form of d-amino-acid oxidase

Abstract

1. 1. The semiquinoid form of d-amino-acid oxidase ( d-amino-acid:O 2 oxidoreductase (deaminating), EC 1.4.3.3) has been isolated by crystallization from the solution obtained by illuminating the purple complex of the enzyme with the substrate, d-alanine, under anaerobic conditions. The occurrence of this species could be explained by the photo-accelerated separation of two electrons shared by the enzyme and the substrate moieties in the complex. 2. 2. Spectroscopic observations indicated that the present crystalline preparation was a mixture of the semiquinoid enzyme-substrate ( d-alanine) complex, and the complex of the semiquinoid enzyme with the products, pyruvate and ammonia. 3. 3. The semiquinoid enzyme could neither be reduced further by the substrate nor be oxidized by the products. It formed blue-colored complexes with benzoate and substituted benzoates, except ortho-substituted ones, without any influence upon its paramagnetic susceptibility. Benzoate could compete with d-alanine and with pyruvate in combining with the semiquinoid enzyme. 4. 4. Although the oxidized enzyme could be converted into its semiquinoid form by irradiation with strong light under anaerobic conditions or by reduction with sodium dithionite, the complex with benzoate, an enzyme-substrate complex model, was very resistant to photo-reduction, and was converted directly into the fully-reduced state by reduction with sodium dithionite.