The active site of flavoprotein oxidases frequently contains two active-site tyrosines, e.g., Y223 and Y238 in D-amino acid oxidases (DAAO) from Rhodotorula gracilis. In the past, these residues were individually mutated to phenylalanine and serine. To further study their role we undertake an interpretation of Y223F-Y238F double mutation. The spectral properties of the double mutant are similar to those of wild-type DAAO, suggesting a minimally altered active site, but its catalytic activity is lower. A first unexpected observation is that for a number of properties the double mutant is more similar to the wild-type DAAO than to the corresponding single-point mutants, e.g., similar kinetic mechanism, substrate specificity, and redox properties. The Y223F-Y238F DAAO also exhibits appreciably weaker binding of the competitive inhibitors' carboxylic acids (synergistic effects), while the K-m values for the substrate D-amino acids are only slightly changed, thus suggesting that the presence of an amino group in the ligand is important for binding to the double mutant. Synergistic effects of the substitutions are also evident on the rate constant of flavin reduction indicating anti-cooperative interaction of the two residues in the hydride transfer process. Our results indicate that Tyr223-OH and Tyr238-OH contribute to the high catalytic efficiency of DAAO allowing the precise alignment required for optimal catalysis. (c) 2005 Elsevier Inc. All rights reserved.