| The reaction mechanism of phenol hydroxylase (E.C.1.14.13.7), from Trichosporon cutaneum, has been shown to proceed via the formation of a 4a-hydroperoxyflavin intermediate, which is converted to a 4a-hydroxy-FAD species upon oxygenating the phenol substrate. The 4a-hydroxy-FAD enzyme, which converts to oxidized enzyme with the loss of H;Using metapyrocatechase to convert the product of phenol hydroxylase to a highly colored species, it was shown that product release appeared concomitant with intermediate III formation in the presence of azide. In the absence of monovalent anion, product release was observed concomitant with the appearance of the first of two oxidized enzyme species.;Isotope effects were found with deuterated resorcinol (1.7-3.7) during the conversion of intermediate II to the 4a-hydroxy-FAD enzyme only. No deuterium isotope effects were found with deuterated phenol, possessing no spectrally visible intermediate II, or deuterated thiophenol, implying the lack of ring proton involvement in S-hydroxylation. Deuterated m-cresol showed only a small deuterium isotope effect (1.2).;Photoreduction of 6-hydroxy-FAD phenol hydroxlase resulted in the transient formation of a semiquinone with a maximum absorbance at 536 nm. The kinetics and spectra of the intermediates formed upon reaction with substrate and molecular oxygen of both reduced native and 6-hydroxy-FAD enzyme were calculated from stopped-flow experiments and compared.;6-Thiocyanato-FAD and 6-mercapto-FAD were substituted for FAD on the enzyme. The anion of the 6-mercapto-FAD is destabilized (pKa = 7.1) relative to free flavin (pK = 5.9). Complexation with phenol decreased the pKa value to 6.3. Comparison of reaction rates of both free 6-mercaptoflavin and 6-mercapto-FAD phenol hydroxylase with thiol-specific reagents revealed that the 6-position is highly accessible to solvent. Accessibility was decreased up to 6-fold in the presence of phenol. |
