Rate-Determining Attack on Substrate Precedes Rieske Cluster Oxidation during Cis-Dihydroxylation by Benzoate Dioxygenase

Abstract

Rieske dearomatizing dioxygenases utilize a Rieske iron-sulfur cluster and a mononuclear Fe(II) located 15 Å across a subunit boundary to catalyze O2-dependent formation of cis-dihydrodiol products from aromatic substrates. During catalysis, O2 binds to the Fe(II) while the substrate bind nearby. Single turnover reactions have shown that one electron from each metal center is required for catalysis. This finding suggested that the reactive intermediate is Fe(III)-(H)peroxo or HO-Fe(V)=O formed by O-O bond scission. Surprisingly, several kinetic phases were observed during the single turnover Rieske cluster oxidation. Here, the Rieske cluster oxidation and product formation steps of a single turnover of benzoate 1,2-dioxygenase are investigated using benzoate and three fluorinated analogs. It is shown that the rate constant for product formation correlates with the reciprocal relaxation time of only the fastest kinetic phase (RRT-1) for each substrate, suggesting that the slower phases are not mechanistically relevant. RRT-1 is strongly dependent on substrate type, suggesting a role for substrate in electron transfer from the Rieske cluster to the mononuclear iron site. This insight, together with the substrate and O2 concentration dependencies of RRT-1, indicates that a reactive species is formed after substrate and O2 binding, but before electron transfer from the Rieske cluster. Computational studies show that RRT-1 is correlated with the electron density at the substrate carbon closest to the Fe(II), consistent with initial electrophilic attack by an Fe(III)-superoxo intermediate. The resulting Fe(III)-peroxo-aryl radical species would then readily accept an electron from the Rieske cluster to complete the cis-dihydroxylation reaction.

NRVS Studies of the Peroxide Shunt Intermediate in a Rieske Dioxygenase and Its Relation to the Native FeII O2 Reaction

“…In these enzymes, a high-spin Fe III –peroxy level intermediate has been trapped in a peroxide shunt reaction, 18 while recent single turnover kinetics data potentially implicate an Fe III –superoxo as an active intermediate in the native O 2 reaction. 19 The Rieske oxygenases catalyze a range of diverse chemistries 20 including monooxygenation, 21 sulfoxidation, 21 and desaturation. 22 Most of the well-studied Rieske oxygenases are dioxygenases (RDOs) that catalyze the cis -dihydroxylation of aromatic rings 23 and are important in bioremediation.…”

“…18 This intermediate, BZDOp, forms the correct cis -dihydroxylated product, 1-carboxy-1,2- cis -dihydroxycyclohexa-3,5-diene (benzoate- cis- diol), but at a much slower rate than in the native O 2 reaction with the NHFe II site 18,19 (5.8 × 10 −4 s −1 vs 190 s −1 , both pseudo first order single-turnover rates). This intermediate has been tentatively assigned as a high-spin Fe III –peroxy species on the basis of Mössbauer spectroscopy, with the binding mode of O 2 , how this relates to the crystallographic intermediates, and protonation state of the O 2 unknown.…”

“…Further, recent single-turnover kinetics on the native O 2 reaction in BZDO indicate that the O 2 -bound NHFe II site reacts with the substrate prior to Rieske center oxidation, which implicates an Fe III –superoxy as the reactive intermediate, rather than an Fe III –(hydro)peroxy. 19 If an Fe III –superoxy is the active intermediate, this raises the question of what drives its formation, as substrate is not coordinated to the Fe, and from reference 42, O 2 binding to an Fe II in a facial triad (without substrate bound) is highly endergonic. Accordingly, O 2 binding in the absence of substrate is not kinetically relevant for either NDO or BZDO based on the single turnover studies.…”

Pathways for Arene Oxidation in Non-Heme Diiron Enzymes: Lessons from Computational Studies on Benzoyl Coenzyme A Epoxidase

“…16 Notwithstanding the above, for benzoate dioxygenase, recent experimental evidence suggests involvement of a species at the Fe(III)-superoxo, instead of peroxo, level in substrate oxidation. 17 Pterin-dependent aromatic hydroxylases, 18 and the few known α-ketoglutarate-dependent aromatic hydroxylases as well (see Figure 1D), 19 employ their cosubstrate and O2 (d1) to form an iron(IV)-oxo active intermediate (d2). Radical attack on the arene yields a substrate radical complexed to iron(III) (d3); migration of a hydrogen atom or a carboxymethyl group then produces a ketone (d4), which eventually regains aromaticity by tautomerization.…”

An aryl dioxygenase shows remarkable double dioxygenation capacity for diverse bis-aryl compounds, provided they are carbocyclic

“…Although the nature of the activated oxygen species attacking the aromatic substrate and, consequently, the reaction mechanism, is not yet entirely clear, most available evidence favours a radical mechanism with consecutive formation of the two novel C-O bonds (Bugg and Ramaswamy 2008; Barry and Challis 2013). In spite of the radical character of the reaction, increased electron density at the primarily attacked carbon appears to enhance reactivity (Barry and Challis 2013; Rivard et al 2015). In agreement with this notion, it was expected that electron-poor aryls are poor and electron-rich aryls are good substrates for ARHDOs (Boyd et al 2008).…”