Studies of a Flavoprotein, Salicylate Hydroxylase

Salicylate hydroxylase has been shown to react with certain analogues, effecting an uncoupling of oxygen reduction from hydroxylation (White-Stevens, R. H., and Kamin, H. (1972) J. Biol. Chem. 247, 2358–2370; (1970) Biochem. Biophys. Res. Commun. 38, 882). The mechnism of action of this enzyme has now been examined by spectrophotometric and stopped flow techniques. Compounds which facilitate DPNH binding and oxidation perturb the absorption spectrum of the enzyme. From this perturbation, an apparent Ks commensurate with catalytic Km values for benzoate and salicylate can be obtained. With salicylate, one salicylate is bound per flavin. Anaerobic reduction with limiting DPNH yields only fully reduced and fully oxidized flavin; semiquinone is formed only with photoreduction in the presence of EDTA. The two flavins act independently. As observed in the stopped flow, formation of enzyme-substrate complex is very rapid; the presence of salicylate facilitates DPNH binding and determines the rate of enzyme reduction to E-FADH2. The catalytic Vmax for DPNH oxidation with several organic compounds appears to be reflected specifically in the rate constants for enzyme reduction. The reoxidation of reduced enzyme by O2 is independent of the nature or even the presence of aromatic compound and is strictly proportional to O2 tension over a wide [O2] range. Depending upon the aromatic molecule, either the rate of reduction of enzyme, the rate of oxidation, or a combination of both can be rate-limiting for catalysis. The hydroxylating species, designated E-FAD-H2O2, must be short lived and can either (a) stoichiometrically hydroxylate a suitable such as salicylate, (b) decompose rapidly and quantitatively to H2O2 if the is totally unsuitable (i.e. benzoate), or (c) hydroxylate with erratic formation of H2O2, for substrates which act in a manner intermediate between the substrate (salicylate) or pseudosubstrate (benzoate) modes.