RESUMEN
The changes in the layered structure of Mg-Al hydrotalcite (Mg/ Al = 2) during heat treatment have been investigated by using in situ XAFS simultaneously at the Mg and Al K-edges. The development of unique in situ instrumentation allowed the coordination environments at both the Mg and Al centers to be monitored as a function of the temperature and heat treatment. The results of this study show that the hydrotalcite structure is highly flexible, and should lead to the further development of hydrotalcites as new solid basic catalysts. Moreover, the Mg and Al cations in the cation layers show different behavior as a function of temperature. The coordination of some octahedral Al ions decreases already at a temperature of 425 K, whereas the coordination about Mg does not show any modification at this temperature. However, hydrotalcite treated at 425 K, followed by cooling down to room temperature resulted in a complete reversal to the original octahedral Al coordination. It is proposed that Al-OH bond breakage occurs at 425 K, without the evolution of H2O. This bond is restored after cooling to room temperature. The actual dehydroxylation of hydrotalcite commences between 425 and 475 K, as indicated by a change in coordination of both the Mg and Al centers. This is accompanied by the evolution of H2O molecules and the changes are hence irreversible without the presence of excess water. Heat treatment at 725 K leads to the development of an MgO-like phase (octahedral Mg) and a mixed octahedral/tetrahedral Al phase. A subsequent rehydration at room temperature entirely restores the original coordination about the Al and Mg centers of hydrotalcite to a distance of 15 A, to which XAFS spectroscopy is sensitive.
RESUMEN
The kinetic mechanism of vanillyl-alcohol oxidase with 4-methylphenol, 4-ethylphenol, 4-propylphenol and their C alpha-deuterated analogs has been studied at pH 7.5 and 25 degrees C. Conversion of 4-methylphenol is extremely slow (0.005 s(-1)) while the enzyme is largely in the reduced form during turnover. 4-Ethylphenol and 4-propylphenol are readily converted while the enzyme is mainly in the oxidized form during turnover. The deuterium kinetic isotope effect for overall catalysis ranges between 7-10 whereas the intrinsic deuterium kinetic isotope effect for flavin reduction ranges over 9-10. With all three 4-alkylphenols, flavin reduction appeared to be a reversible process with the rate of reduction being in the same range as the rate for the reverse reaction. During the reductive half-reaction of vanillyl-alcohol oxidase with 4-ethylphenol and 4-propylphenol, a transient intermediate is formed with an absorbance maximum at 330 nm. This intermediate has been tentatively identified as the p-quinone methide of the aromatic substrate in complex with reduced enzyme. It is concluded that vanillyl-alcohol oxidase catalysis with 4-ethylphenol and 4-propylphenol favors an ordered sequential binding mechanism in which the rate of flavin reduction determines the turnover rate while the reduced enzyme-p-quinone methide binary complex rapidly reacts with dioxygen. During the reaction of vanillyl-alcohol oxidase with 4-methylphenol, a fluorescent enzyme species is stabilized. Based on its spectal characteristics and crystallographic data [Mattevi, A., Fraaije, M. W., Mozzarelli, A., Olivi, L., Coda, A. & van Berkel, W. J. H. (1997) Structure 5, 907-920], it is proposed that this species represents a covalent 5-(4'-hydroxybenzyl)-FAD adduct. With 4-ethylphenol and 4-propylphenol, similar N5 flavin adducts may be formed but their rate of formation is too slow to be of catalytic relevance.