Chemistry of the catalytic conversion of phthalate into its cis-dihydrodiol during the reaction of oxygen with the reduced form of phthalate dioxygenase

The phthalate dioxygenase system, a Rieske non-heme iron dioxygenase, catalyzes the dihydroxylation of phthalate to form the 4,5-dihydro-cis-dihydrodiol of phthalate (DHD). It has two components: phthalate dioxygenase (PDO), a multimer with one Rieske-type [2Fe-2S] and one mononuclear Fell center per monomer, and a reductase (PDR) that contains flavin mononucleotide (FMN) and a plant-type ferredoxin [2Fe-2S] center. This work shows that product formation in steady-state reactions is tightly coupled to electron delivery, with 1 dihydrodiol (DHD) of phthalate formed for every 2 electrons delivered from NADH. However, in reactions of reduced PDO with O-2, only about 0.5 DHD is formed per Rieske center that becomes oxidized. Although the product forms rapidly, its release from PDO is slow in these reactions with oxygen that do not include reductase and NADH. EPR data show that, at the completion of the oxidation, iron in the mononuclear center remains in the ferrous state. In contrast, naphthalene dioxygenase (NDO) [Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953] and benzoate dioxygenase (BZDO) [Wolfe, M. D., Altier, D. J., Stubna, A., Popescu, C. V., Munck, E., and Lipscomb, J. D. (2002) Biochemistry, 41, 9611-9626], related Rieske non-heme iron dioxygenases, form 1 DHD per Rieske center oxidized, and the mononuclear center iron ends up ferric. Thus, both electrons from reduced NDO and BZDO monomers are used to form the product, whereas only the reduced Rieske centers in PDO become oxidized during production of DHD. This emphasizes the importance of PDO subunit interaction in catalysis. Electron redistribution was practically unaffected by the presence of oxidized PDR. A scheme is presented that emphasizes some of the differences in the mechanisms involved in substrate hydroxylation employed by PDO and either NDO or BZDO.