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

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 (PDO(red)) that lacks Fe(II) at the mononuclear metal site (PDO-APO) reacts slowly with O(2) (1.4 x 10(-3) s(-1) at 125 microM O(2) and 22 degrees C), presumably in a direct reaction with the Rieske center. Binding of phthalate and/or PDR(ox) to reduced PDO-APO increases the reactivity of the Rieske center with O(2). When no PDR or phthalate is present, the oxidation of the Rieske center in native PDO(red) [which contains Fe(II) at the mononuclear site] occurs in two phases (approximately 1 and 0.1 s(-1) at 125 mM O(2), 23 degrees C), both much faster than in the absence of Fe(II), presumably because in this case O(2) reacts at the mononuclear Fe(II). Addition of PDR(ox) to native PDO(red) 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 PDR(ox) and phthalate present, most of the PDO(red) (approximately 80-85%) oxidizes at 42 s(-1), with the remaining oxidizing at approximately 5 s(-1). Thus, the binding of phthalate or PDR(ox) to PDO(red) each results in greater reactivity of PDO with O(2). 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.

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

“…Plasmids containing WT PDO and D178N and D178A variants genes were constructed and the proteins were expressed and purified as described previously (14). Concentrations of enzymes were determined spectrophotometrically using Δε 575 = 2.38 mM −1 cm −1 and Δε 466 = 17.54 mM −1 cm −1 for the extinction coefficient differences between oxidized and reduced PDO and PDR, respectively.…”

“…Concentrations of enzymes were determined spectrophotometrically using Δε 575 = 2.38 mM −1 cm −1 and Δε 466 = 17.54 mM −1 cm −1 for the extinction coefficient differences between oxidized and reduced PDO and PDR, respectively. PDO activity was determined in steady-state assays as described previously (14). When necessary, Fe(NH 4 ) 2 (SO 4 ) 2 (FAS), prepared as an anaerobic solution of known concentration, was added.…”

“…When necessary, Fe(NH 4 ) 2 (SO 4 ) 2 (FAS), prepared as an anaerobic solution of known concentration, was added. PDO-APO, which lacks iron in the mononuclear center, and a less stringent preparation (PDO-APO 20 ), which contained about 2.2 Fe/monomer, were prepared as described previously (14). Iron content in PDO was determined by Inductively Coupled Plasma High Resolution Mass Spectrometry (ICP) using a Finnegan Element Instrument from Thermo Finnegan Co.…”

“…Similar to NDO (9) and BZDO (28), when the Rieske center in PDO is reduced, NO binds to the mononuclear iron only when substrate is present (14). However, when the Rieske center is oxidized, NO can bind both in the presence and in the absence of the substrate (14). Such gating of NO (and by extension, possibly O 2 ) binding by the redox state of the Rieske center and the availability of the substrate appears to be a common feature of this family of Rieske dioxygenases.…”

“…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). Using NO as a limited model for oxygen binding at the Fe(II) of the mononuclear center , it was found that NO can bind to the mononuclear Fe(II) if the Rieske center is oxidized, but not when it is reduced, unless substrate is also bound at the mononuclear center (9,14). Thus, there is important communication between the Rieske and mononuclear sites, and this communication has been proposed to be due to His-104, His-213, and Asp-205 in NDO (9).…”