Wavelength (nm)

Fig. 5. Reduction of D170S by the xanthine-xanthine oxidase method. Curve 1, quinone form; curve 2, semiquinone anionic form; curve 3, hydroquinone form of D170S. [Data from R. H. H. van den Heuvel, M. W. Fraaije, A. Mattevi, and W. J. H. van Berkel, J. Biol. Chem. 275, 14799 (2000).]

and the orientation of the negative charge near flavin N5 are crucial for the high redox potential of the flavin in VAO, and thus for the oxidative power of the enzyme. The behavior of the D170S mutant in the xanthine oxidase-mediated reduction is different from wild-type VAO, as this mutant highly stabilizes the red flavin semiquinone anion with a typical absorption maximum at 385 nm (Fig. 5). This thermodynamic stabilization of the semiquinone is different from kinetic substrate-mediated reduction experiments, in which no semiquinone formation is observed in the stopped-flow time scale.33

Redox potential determinations of flavin derivatives have revealed that modifications at the 8a position of the isoalloxazine ring result in increased redox potentials by 50-60 mV37'38 and that ionization of the imidazole of 8a-N-imidazolylflavins results in changed redox properties.39 To investigate the effect of the 8a-(iV3-histidyl)-flavin linkage in VAO, we substituted His-422, the residue to which the flavin is bound to the protein, for alanine.11 The produced VAO mutant H422A binds the flavin tightly but noncovalently. The H422A mutation does not have any significant effects on the structure of the enzyme, but lowers the turnover rate with the physiological substrate 4-(methoxymethyl)phenol by one order of magnitude. The estimation of the redox potential of H422A revealed a 120-mV lower potential than for wild-type VAO. As Ala-422 is relatively far from the catalytic center of VAO, additional effects on the kinetics of the enzyme are unlikely. Therefore, the lower activity of the enzyme must be fully attributed to the decreased redox potential of the flavin.11 This is the first report in which it has been

37 D. E. Edmondson and T. P. Singer, J. Biol. Chem. 248, 8144 (1973).

38 D. E. Edmondson and R. De Francesco, in "Chemistry and Biochemistry of Flavoenzymes" (F. Mueller, ed.), Vol. I, p. 73. CRC Press, Boca Raton, FL, 1991.

39 G. Williamson and D. E. Edmondson, Biochemistry 24, 7918 (1985).

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