Covalent flavinylation is essential for efficient redox catalysis in vanillyl-alcohol oxidase

By mutating the target residue of covalent flavinylation in vanillyl-alcoho l oxidase, the functional role of the histidyl-FAD bond was studied. Three His(422) mutants (H422A, H422T, and H422C) were purified, which all contain ed tightly but noncovalently bound FAD. Steady state kinetics revealed that the mutants have retained enzyme activity, although the turnover rates hav e decreased by 1 order of magnitude. Stopped-flow analysis showed that the H422A mutant is still able to form a stable binary complex of reduced enzym e and a quinone methide product intermediate, a crucial step during vanilly l-alcohol oxidase-mediated catalysis, The only significant change in the ca talytic cycle of the H422A mutant is a marked decrease in reduction rate. R edox potentials of both wild type and H422A vanillyl-alcohol oxidase have b een determined. During reduction of H422A, a large portion of the neutral f lavin semiquinone is observed. Using suitable reference dyes, the redox pot entials for the two one-electron couples have been determined: -17 and -113 mV. Reduction of wild type enzyme did not result in any formation of flavi n semiquinone and revealed a remarkably high redox potential of +55 mV, The marked decrease in redox potential caused by the missing covalent histidyl -FAD bond is reflected in the reduced rate of substrate-mediated flavin red uction limiting the turnover rate. Elucidation of the crystal structure of the H422A mutant established that d eletion of the histidyl-FAD bond did not result in any significant structur al changes. These results clearly indicate that covalent interaction of the isoalloxazine ring with the protein moiety can markedly increase the redox potential of the flavin cofactor, thereby facilitating redox catalysis, Th us, formation of a histidyl-EAD bond in specific flavoenzymes might have ev olved as a way to contribute to the enhancement of their oxidative power.