MECHANISM OF P-HYDROXYPHENYLACETATE-3-HYDROXYLASE - A 2-PROTEIN ENZYME

p-Hydroxyphenylacetate-3-hydroxylase purified from Pseudomonas putida is a two-protein enzyme requiring a flavoprotein and a coupling protei n for productive hydroxylation (Arunachalam, U., Massey, V., and Vaidy anathan, C. S. (1992) J. Biol. Chem. 267,25848-25855). This paper pres ents information on the mechanism of the enzyme from absorbance and fl uorescence stopped-flow studies. The reduction of the substrate-free f lavoprotein by NADH was slow and was not altered by the presence of th e coupling protein. In contrast, the coupling protein has a dramatic e ffect in the oxidative half-reaction. The flavoprotein when present al one, both in the absence and presence of the aromatic substrate, react s in a second-order fashion with oxygen to form oxidized flavoprotein, with no indication of flavin-oxygen intermediates. However, an interm ediate identified as the C4a-flavin hydroperoxide is stabilized when t he flavoprotein-coupling protein complex reacts with oxygen in the abs ence of the aromatic substrate, p-hydroxyphenylacetate, and at least t hree flavin-oxygen intermediates, attributed to the anionic (I) and pr otonated (I) forms of the flavin hydroperoxide and the hydroxyflavin (III), are formed in the oxidative half-reaction in the presence of th e aromatic substrate. A reaction mechanism for the two-protein complex is proposed in which the aromatic substrate has little effect on the rate of reduction of the enzyme flavin but has strict control in the o xidative half-reaction. In this phase the flavin hydroperoxide is rema rkably stable in the absence of the substrate but disappears rapidly u pon encountering the aromatic substrate.