Salicylate 5-Hydroxylase: Intermediates in Aromatic Hydroxylation by a Rieske Monooxygenase

Abstract

Rieske oxygenases (ROs) catalyze a large range of oxidative chemistry. We have shown that cis-dihydrodiol-forming Rieske dioxygenases first react with their aromatic substrates via an active site nonheme Fe­(III)-superoxide; electron transfer from the Rieske cluster then completes the product-forming reaction. Alternatively, two-electron-reduced Fe­(III)–peroxo or hydroxo–Fe­(V)–oxo activated oxygen intermediates are possible and may be utilized by other ROs to expand the catalytic range. Here, the reaction of a Rieske monooxygenase, salicylate 5-hydroxylase, that does not form a cis-dihydrodiol is examined. Single-turnover kinetic studies show fast binding of salicylate and O2. Transfer of the Rieske electron required to form the gentisate product occurs through bonds over ∼12 Å and must also be very fast. However, the observed rate constant for this reaction is much slower than expected and sensitive to substrate type. This suggests that initial reaction with salicylate occurs using the same Fe­(III)–superoxo-level intermediate as Rieske dioxygenases and that this reaction limits the observed rate of electron transfer. A transient intermediate (λmax = 700 nm) with an electron paramagnetic resonance (EPR) at g = 4.3 is observed after the product is formed in the active site. The use of 17O2 (I = 5/2) results in hyperfine broadening of the g = 4.3 signal, showing that gentisate binds to the mononuclear iron via its C5-OH in the intermediate. The chromophore and EPR signal allow study of product release in the catalytic cycle. Comparison of the kinetics of single- and multiple-turnover reactions shows that re-reduction of the metal centers accelerates product release ∼300-fold, providing insight into the regulatory mechanism of ROs.

Structural basis of carnitine monooxygenase CntA substrate specificity, inhibition, and intersubunit electron transfer

“…2 E ). As it is known that substrate binding can be required for the activation of the catalytic mononuclear Fe center to initiate catalysis ( 42 , 43 ), the substrate (carnitine) was added to the reaction and the EPR spectrum was monitored ( Fig. 2 C , cyan trace).…”

“…None of the E205 mutants that we generated in this study were active ( Figure 2D), with no major perturbations to the overall secondary structure as observed by circular dichroism (Figure 2E). As it is known that substrate-binding can be required for the activation of the catalytic mononuclear Fe centre to initiate catalysis (42)(43), the substrate (carnitine) was added to the reaction and the EPR spectrum was monitored ( Figure 2C, cyan trace). The spectrum shows no EPR signals at low magnetic fields, ruling out the presence of high-spin (S = 5/2 or 3/2) ferric species in the sample.…”

Structural basis for divergent C–H hydroxylation selectivity in two Rieske oxygenases

“…To accomplish selective modification of inert C-H bonds, these enzyme classes employ molecular oxygen to generate high-valent iron intermediates, which activate a substrate for hydroxylation through hydrogen atom abstraction [7][8][9][10][11] . Examples include the Fe(IV)-oxo and Fe (III)-hydroperoxo intermediates employed by heme-containing systems 12,13 and the Fe(III)-superoxo 14 , Fe(IV)-oxo, or proposed Fe(V)-oxo species used by non-heme systems 7 . In the Rieske oxygenase class, work to date has been conducted on a small sample of enzymes, specifically those that perform dioxygenation of aromatic rings.…”

Spectroscopic Characterisation of the Naphthalene Dioxygenase from Rhodococcus sp. Strain NCIMB12038

“…Our data are in agreement with those previously reported for NDO from Pseudomonas sp. [7,15]. Proteins containing the [2Fe-2S] clusters exhibit this characteristic EPR signature, formed of one g -value just above g = 2 (range: 2.01–2.05) and two g -values below g = 2 typically between 1.85 and 1.95.…”

“…Recently [15] it has been reported that the Fe(II) mononuclear iron and the one-electron reduced Rieske cluster are able to catalyse the dihydroxylation of naphthalene in the absence of NDR and NDF in a single turnover reaction [7]. Naphthalene and molecular oxygen react with NDO to form an amount of product that is stoichiometric with the concentration of the mononuclear Fe(II) sites.…”