Rates of the Phthalate Dioxygenase Reaction with Oxygen Are Dramatically Increased by Interactions with Phthalate and Phthalate Oxygenase Reductase

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Abstract
The phthalate dioxygenase system, which catalyzes the dihydroxylation of phthalate to form its cis-dihydrodiol (DHD), has two components: phthalate dioxygenase (PDO), a multimer with one Rieske-type [2Fe-2S] and one Fe(II) center per monomer, and phthalate dioxygenase reductase (PDR), which contains flavin mononucleotide (FMN) and a plant-like ferredoxin [2Fe-2S] center. PDR is responsible for transferring electrons from NADH to the Rieske center of PDO, and the Rieske center supplies electrons to the mononuclear center for the oxygenation of substrate. Reduced PDO (PDOred) that lacks Fe(II) at the mononuclear metal site (PDO-APO) reacts slowly with O2 (1.4 × 10-3 s-1 at 125 μM O2 and 22 °C), presumably in a direct reaction with the Rieske center. Binding of phthalate and/or PDRox to reduced PDO-APO increases the reactivity of the Rieske center with O2. When no PDR or phthalate is present, the oxidation of the Rieske center in native PDOred [which contains Fe(II) at the mononuclear site] occurs in two phases (∼1 and 0.1 s-1 at 125 mM O2, 23 °C), both much faster than in the absence of Fe(II), presumably because in this case O2 reacts at the mononuclear Fe(II). Addition of PDRox to native PDOred resulted in a large fraction of the Rieske center being oxidized at 5 s-1, and the addition of phthalate resulted in about 70% of the reaction proceeding at 42 s-1. With both PDRox and phthalate present, most of the PDOred (approximately 80−85%) oxidizes at 42 s-1, with the remaining oxidizing at ∼5 s-1. Thus, the binding of phthalate or PDRox to PDOred each results in greater reactivity of PDO with O2. The presence of both the substrate and PDR was synergistic, making PDO fully catalytically active. A model that explains the observed effects is presented and discussed in terms of PDO subunit cooperativity. It is proposed that, during oxidation of reduced PDO, each of two Rieske centers on separate subunits transfers an electron to the Fe(II) mononuclear center on a third subunit. This explanation is consistent with the observed multiphasic kinetics of the oxidation of the Rieske center and is being further tested by product analysis experiments.