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Fig. 6. Reduction of H422A by the xanthine-xanthine oxidase method. Curve 1, quinone form; curve 2, semiquinone neutral form; curve 3, hydroquinone form of H422A. [Data from M. W. Fraaije, R. H. H. van den Heuvel, W. J. H. van Berkel, and A. Mattevi, J. Biol. Chem. 274,35514 (1999).]

325 425 525 625

Wavelength (nm)

Fig. 6. Reduction of H422A by the xanthine-xanthine oxidase method. Curve 1, quinone form; curve 2, semiquinone neutral form; curve 3, hydroquinone form of H422A. [Data from M. W. Fraaije, R. H. H. van den Heuvel, W. J. H. van Berkel, and A. Mattevi, J. Biol. Chem. 274,35514 (1999).]

demonstrated that a covalent protein-flavin bond raises the redox potential of the flavin, and, therefore, the oxidative power of the flavoenzyme. Interestingly, the xanthine oxidase-mediated reduction of H422A occurs in two discrete one-electron reduction steps. First, a blue neutral flavin semiquinone species is formed, which is further reduced to the flavin hydroquinone (Fig. 6). Thus, the H422A mutation shifts the piTof the flavin semiquinone to a higher value compared with the D170S mutation, resulting in the neutral form of the flavin semiquinone at pH 7.5.

Conclusions

In the described research we have investigated the redox properties of the flavin prosthetic group in VAO. Site-directed mutagenesis studies have revealed that the exceptional redox properties of VAO are, at least in part, due to two rare structural determinants: the negative charge of Asp-170 near the flavin N5 atom and the covalent histidyl linkage between the protein and the flavin. The negative charge at hydrogen bond distance from the flavin N5 atom (3.5 A) stabilizes the reduced form of the cofactor, thus facilitating the oxidation of substrates. Except for glycolate oxidase,36 none of the other flavoenzymes with known three-dimensional structure harbor a hydrogen bond acceptor near flavin N5.35 In agreement with earlier suggestions,9 a covalent linkage between the protein and the isoalloxazine ring of the flavin can markedly increase the redox potential of the flavin prosthetic group.11 This indicates that the histidyl-flavin bond in certain flavoproteins has evolved as a tool for raising the oxidative power of the flavin cofactor.

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