Studies of a Flavoprotein, Salicylate Hydroxylase

A flavoprotein, salicylate hydroxylase (salicylate, DPNH : oxygen oxidoreductase (I -hydroxylation, l-decarboxylating)), has been induced and isolated from a soil bacterium grown on salicylate as sole carbon source. In contrast to a similar enzyme studied by YAMAMOTO, S., KATAGIRI, M., MAENO, H., AND HAYAISHI, 0. ((1965) J. Bid, Chem. 240, 3408) this enzyme is dimeric with two subunits and two FAD per 91,000 molecular weight (rather than one FAD per 57,000). When benzoate is substituted for salicylate, DPNH is oxidized with the same Max as with salicylate but with higher Km for both benzoate and DPNH. With salicylate, the reaction products are catechol and HZO; with benzoate, the benzoate is unchanged, but H2U2 is formed stoichiometrically with DPNH oxidized. Both salicylate and benzoate facilitate DPNH binding. Benzoate binds at the salicylate site, competitively inhibiting salicylate hydroxylation, and permitting DPNH binding and oxidation. But since benzoate, a “pseudosubstrate,” cannot be hydroxylated, the oxygen utilized decomposes to H202, and oxygen reduction is considered as C‘uncoupled” from hydroxylation. A search for possible active intermediates in the reduction of O2 to Hz02 by the uncoupled reaction has failed to yield evidence for any oxygen radical species. An examination of various substituted benzoates and salicylates has revealed a range of behavior intermediate between the “substrate” (salicylate) and pseudosubstrate (benzoate) modes. These compounds are hydroxylated, (in several cases more rapidly than salicylate), but some of the oxygen utilized is diverted to H&. These compounds are bound to enzyme with a K, higher than the inducer, salicylate, and also facilitate DPNH binding, but less effectively than does salicylate. Other properties of this enzyme, including inhibition by some monovalent anions, are described.