On the reaction mechanism of phenol hydroxylase. New information obtained by correlation of fluorescence and absorbance stopped flow studies.

Steps in the hydroxylation pathway of the flavoprotein phenol hydroxylase with resorcinol as substrate have been studied by a combination of fluorescence and absorbance stopped flow techniques. In the presence of azide, a series of highly fluorescent oxygenated flavin intermediates has been observed, corresponding to those previously detected by absorbance measurements (Detmer, K., and Massey, V. (1985) J. Biol. Chem. 260, 5998-6005). In addition, yet another intermediate has been found as the immediate product of the reaction of the reduced enzyme with O2. This new species is non-fluorescent in the presence of azide, but fluorescent in the absence of monovalent anions and had escaped detection in previous absorbance studies because of the similarity in its rates of formation and conversion to the next intermediate and similarity in their spectra. These two early intermediates are tentatively identified as the anionic and protonated species of the flavin C4a-hydroperoxide or, alternatively, as two conformationally different forms of the enzyme hydroperoxide. The next intermediate, previously referred to as intermediate II, is also highly fluorescent and so is considered unlikely to be due to a complex of a flavin alkoxyl radical and a substituted cyclohexadienyl radical, as proposed by Anderson et al. (Anderson, R. F., Patel, K. B., and Stratford, M. R. L. (1990) J. Biol. Chem. 265, 1952-1957). The conversion of intermediate II to the next intermediate, intermediate III (the C4a-hydroxyflavin), is characterized by a large substrate deuterium isotope effect in the 320-390 nm range, but not by fluorescence or by absorbance at wavelengths > 400 nm. This is ascribed to dissociation from the enzyme of a cyclohexadienone product, leaving the enzyme in its C4a-hydroxyflavin form. The latter eliminates H2O to re-form oxidized flavin, but in a competing reaction, in the presence of excess substrate, forms a very stable complex, which decays orders of magnitude more slowly than the uncomplexed enzyme.