Redox potentials and their pH dependence of D-amino-acid oxidase of Rhodotorula gracilis and Trigonopsis variabilis.
The redox potentials and pH characteristics of D-amino-acid oxidase (EC 1.4.3.3; DAAO) from the yeast Rhodotorula gracilis and Trigonopsis variabilis were measured in the pH range 6.5-8.5 at 15 degrees C. In the free enzyme form, the anionic red semiquinone is quantitatively formed in both DAAOs, indicating that a two single-electron transfer mechanism is active. The semiquinone species is also thermodynamically stable, as indicated by the large separation of the single-electron transfer potentials. The first electron potential is pH-independent, while the second electron transfer is pH-dependent exhibiting a approximately -60 mV/pH unit slope, consistent with a one-electron/one-proton transfer. In the presence of the substrate analogue benzoate, the two-electron transfer is the thermodynamically favoured process for both DAAOs, with only a quantitative difference in the stabilization of the anionic semiquinone. Clearly binding of the substrate (or substrate analogue) modulates the redox properties of the two enzymes. In both cases, in the presence and absence of benzoate, the slope of Em vs. pH (-30 mV/pH unit) corresponds to an overall two-electron/one-proton transfer in the reduction to yield the anionic reduced flavin. This behaviour is similar to that reported for DAAO from pig kidney. The differences in potentials and the stability of the semiquinone intermediate measured for the three DAAOs probably stem from different isoalloxazine environments. In the case of R. gracilis DAAO, the low stability of the semiquinone form in the DAAO-benzoate complex can be explained by the shift in position of the side chain of Arg285 following substrate analogue binding.
Pollegioni
L
L
Department of Structural and Functional Biology, University of Insubria, Varese, Italy.
Porrini
D
D
Molla
G
G
Pilone
M S
MS
D001203:Ascomycota / Q000201:enzymology*; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D012248:Rhodotorula / Q000201:enzymology*; D013053:Spectrophotometry
D003605:D-Amino-Acid Oxidase