Catalytic mechanism of the oxidative demethylation of 4-(methoxymethyl)phenol by vanillyl-alcohol oxidase - Evidence for formation of a p-quinone methide intermediate

The catalytic mechanism for the oxidative demethylation of 4-(methoxymethyl)phenol by the covalent flavoprotein vanillyl-alcohol oxidase was studied, Using (H2O)-O-18, it was found that the carbonylic oxygen atom from the product 4-hydroxybenzaldehyde originates from a water molecule. Oxidation of vanillyl alcohol did not result in any incorporation of O-18. Enzyme-monitored turnover experiments revealed that for both substrates a process involving flavin reduction is rate determining, During anaerobic reduction of vanillyl-alcohol oxidase by 4-(methoxymethyl)phenol, a relatively stable spectral intermediate is formed, Deconvolution of its spectral characteristics showed a typical ps-independent absorption maximum at 364 nm (epsilon(364 nm) = 46 mM(-1) cm(-1)). A similar transient species was observed upon anaerobic reduction by vanillyl alcohol. The rate of flavin reduction and synchronous intermediate formation by 4-(methoxymethyl)phenol is 3.3 s(-1) and is fast enough to account for turnover (3.1 s(-1)). The anaerobic decay of the intermediate was too slow (0.01 s(-1)) to be of catalytical relevance, The reduced binary complex is rapidly reoxidized (1.5 x 10(5) M-1 s(-1)) and is accompanied with formation and release of product. Oxidation of free-reduced enzyme is an even faster process (3.1 x 10(5) M-1 s(-1)). The kinetic data for the oxidative demethylation of 4-(methoxymethyl)phenol are in accordance with a ternary complex mechanism in which the reduction rate is rate-limiting. It is proposed that, upon reduction, a binary complex is produced composed of the p-quinone methide of 4-(methoxymethyl)phenol and reduced enzyme.