Chemistry of the Catalytic Conversion of Phthalate into Its cis-Dihydrodiol during the Reaction of Oxygen with the Reduced Form of Phthalate Dioxygenase

Abstract

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 Fe(II) 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.

Similar enzymes, different structures: Phthalate dioxygenase is an α3α3 stacked hexamer, not an α3β3 trimer like “normal” Rieske oxygenases

“…Such an arrangement can be formally expressed as α 3 α 3 . This arrangement still allowed for two Rieske and two mononuclear centers to be in proximity to each other, and account for our earlier findings on the stoichiometry of DHD formation during catalysis [8]. Because efforts to obtain the crystal structure of PDO so far have not been successful, we have attempted to clarify the question of the multimeric structure of PDO by other methods.…”

“…On the other hand, E110, E210, and E220 would produce two molecules of product, and E111, E211, E221, and E222 would produce three molecules of DHD per 6 Rieske centers oxidized. Data presented in the table are based on the additional assumption vii) that all mononuclear centers are active, which is true at least for the WT enzyme used in both this study and previously [8]. Combined, the amounts of DHD produced for each possible distribution of Fe (II) among the iron mononuclear centers and the fractional content of these species determine the amount of DHD PDO hexamer would produce at each level of extrageneous free iron.…”

“…PDO was shown to lack β subunits, and based on earlier gel filtration data, was believed to be an α 4 tetramer of 50 kDa monomers (∼ 200 kDa for the multimer) [19;20]. Although the tetramer model, by placing two Rieske centers and two mononuclear centers close to each other, allowed for a consistent interpretation of experimental data for kinetics and product formation [8], the α 4 multimer was clearly dissimilar to the α 3 β 3 multimeric arrangement found in other known Rieske dioxygenases. It seemed surprising that the enzymes from the same family, which perform essentially similar hydroxylation reactions, would differ so much in the multimeric structural arrangements.…”

The “Bridging” Aspartate 178 in Phthalate Dioxygenase Facilitates Interactions between the Rieske Center and the Iron(II)−Mononuclear Center

“…Oxygen reacts poorly with the ferrous mononuclear center if the Rieske center is not reduced, and the presence of substrate greatly enhances oxygen reactivity (9). The binding of NO to ferrous sites has been previously used as a model for oxygen binding in heme as well as in Rieske proteins (9)(10)(11). Certain caveats should be noted concerning the exact geometry of binding in the non-heme enzymes; for example, while the binding of oxygen in NDO was shown to be side-on to the mononuclear iron (12), the binding to NO was shown to occur in an end-on fashion (13).…”

Carbon source-dependent modulation of NADP-glutamate dehydrogenases in isophthalate-degrading Pseudomonas aeruginosa strain PP4, Pseudomonas strain PPD and Acinetobacter lwoffii strain ISP4

“…There are very few reports that describe the utilization of all three phthalate isomers as the carbon source by a single microbial strain (Vamsee-Krishna et al, 2006;Vamsee-Krishna & Phale, 2008;Wang et al, 1995). The first key step involved in the bacterial degradation of phthalate isomers is the double hydroxylation of the aromatic ring by the respective phthalate dioxygenase to yield a cis-dihydrodiol intermediate, which is further metabolized to aliphatic intermediates of the central carbon cycle via 3,4-dihydroxybenzoate (3,4-DHB) (Ballou & Batie, 1988;Batie et al, 1987;Schlafli et al, 1994;Tarasev & Ballou, 2005;Vamsee-Krishna et al, 2006).…”