Asp-170 is crucial for the redox properties of vanillyl-alcohol oxidase

Vanillyl-alcohol oxidase is a flavoprotein containing a covalent flavin tha t catalyzes the oxidation of 4-(methogymethyl)phenol to 4-hydroxybenzaldehy de. The reaction proceeds through the formation of a p-quinone methide inte rmediate, after which, water addition takes place. Asp-170, located near th e N5-atom of the flavin, has been proposed to act as an active site base. T o test this hypothesis, we have addressed the properties of D170E, D170S, D 170A, and D170N variants. Spectral and fluorescence analysis, together with the crystal structure of D170S, suggests that the Asp-170 replacements do not induce major structural changes. However, in D170A and D170N, 50 and 10 0%, respectively, of the flavin is non-covalently bound. Kinetic characteri zation of the vanillyl-alcohol oxidase variants revealed that Asp170 is req uired for catalysis, D170E is 50-fold less active, and the other Asp-170 va riants are about 10(3)-fold less active than wild type enzyme. Impaired cat alysis of the Asp-170 variants is caused by slow flavin reduction. Furtherm ore, the mutant proteins have lost the capability of forming a stable compl ex between reduced enzyme and the p-quinone methide intermediate. The redox midpoint potentials in D170E (+6 mV) and D170S (-91 mV) are considerably d ecreased compared with wild type vanillyl-alcohol oxidase (+55 mV), This su pports the idea that Asp-170 interacts with the protonated N5-atom of the r educed cofactor, thus increasing the FAD redox potential. Taken together, w e conclude that Asp170 is involved in the process of autocatalytic flavinyl ation and is crucial for efficient redox catalysis.