Intradiol Dioxygenases — The Key Enzymes in Xenobiotics Degradation (original) (raw)
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ACS Catalysis
Rieske dioxygenases catalyze the initial steps in the hydroxylation of aromatic compounds and are critical for the metabolism of xenobiotic substances. Because substrates do not bind to the mononuclear non-heme Fe II center, elementary steps leading to O 2 activation and substrate hydroxylation are difficult to delineate, thus making it challenging to rationalize divergent observations on enzyme mechanisms, reactivity, and substrate specificity. Here, we show for nitrobenzene dioxygenase, a Rieske dioxygenase capable of transforming nitroarenes to nitrite and substituted catechols, that unproductive O 2 activation with the release of the unreacted substrate and reactive oxygen species represents an important path in the catalytic cycle. Through correlation of O 2 uncoupling for a series of substituted nitroaromatic compounds with 18 O and 13 C kinetic isotope effects of dissolved O 2 and aromatic substrates, respectively, we show that O 2 uncoupling occurs after the rate-limiting formation of Fe III-(hydro)peroxo species from which substrates are hydroxylated. Substituent effects on the extent of O 2 uncoupling suggest that the positioning of the substrate in the active site rather than the susceptibility of the substrate for attack by electrophilic oxygen species is responsible for unproductive O 2 uncoupling. The proposed catalytic cycle provides a mechanistic basis for assessing the very different efficiencies of substrate hydroxylation vs unproductive O 2 activation and generation of reactive oxygen species in reactions catalyzed by Rieske dioxygenases.
FEBS Letters, 1998
The intradiol cleaving dioxygenases hydroxyquinol 1,2-dioxygenase (HQ1,2O) from Nocardiodes simplex 3E, chlorocatechol 1,2-dioxygenase (ClC1,2O) from Rhodococcus erythropolis 1CP, and their anaerobic substrate adducts (hydroxyquinol-HQ1,2O and 4-chlorocatechol-ClC1,2O) have been characterized through X-ray absorption spectroscopy. In both enzymes the iron(III) is pentacoordinated and the distance distribution inside the Fe(III) first coordination shell is close to that already found in the extensively characterized protocatechuate 3,4-dioxygenase. The coordination number and the bond lengths are not significantly affected by the substrate binding. Therefore it is confirmed that the displacement of a protein donor upon substrate binding has to be considered a general step valid for all intradiol dioxygenases.
Biochemistry, 2006
In Sphingomonas CHY-1, a single ring-hydroxylating dioxygenase is responsible for the initial attack of a range of polycyclic aromatic hydrocarbons (PAHs) composed of up to five rings. The components of this enzyme were separately purified and characterized. The oxygenase component (ht-PhnI) was shown to contain one Rieske-type [2Fe-2S] cluster and one mononuclear Fe center per alpha subunit, based on EPR measurements and iron assay. Steady-state kinetic measurements revealed that the enzyme had a relatively low apparent Michaelis constant for naphthalene (K m = 0.92 ± 0.15 µM), and an apparent specificity constant of 2.0 ± 0.3 µM-1 s-1. Naphthalene was converted to the corresponding 1,2dihydrodiol with stoichiometric oxidation of NADH. On the other hand, the oxidation of eight other PAHs occurred at slower rates, and with coupling efficiencies that decreased with the enzyme reaction rate. Uncoupling was associated with hydrogen peroxide formation, which is potentially deleterious to cells and might inhibit PAH degradation. In single turnover reactions, ht-PhnI alone catalyzed PAH hydroxylation at a faster rate in the presence of organic solvent, suggesting that the transfer of substrate to the active site is a limiting factor. The four-ring PAHs chrysene and benz[a]anthracene were subjected to a double ringdihydroxylation, giving rise to the formation of a significant proportion of bis-cisdihydrodiols. In addition, the dihydroxylation of benz[a]anthracene yielded three dihydrodiols, the enzyme showing a preference for carbons in positions 1,2 and 10,11. This is the first characterization of a dioxygenase able to dihydroxylate PAHs made up of four and five rings. 4 Ring-hydroxylating dioxygenases (RHDs) are widely spread bacterial enzymes that play a critical role in the biological degradation of a large array of aromatic compounds, including polycyclic aromatic hydrocarbons (PAHs)(1, 2). RHDs catalyze the initial oxidation step of such compounds, which consists in the hydroxylation of two adjacent carbon atoms of the aromatic ring, thus generating a cis-dihydrodiol. This reaction converts hydrophobic, often toxic, molecules, into more hydrophilic products, allowing for their subsequent metabolism by other bacterial enzymes. Some RHDs were found to attack highly recalcitrant environmental pollutants, including dibenzo p-dioxin (3, 4), polychlorobiphenyls (5), and PAHs (6-8), thus promoting studies on this type of enzymes with the ultimate goal of improving bioremediation processes (2, 9). RHDs are multi-component enzymes, generally composed of a NADH-oxidoreductase, a ferredoxin and an oxygenase component that contains the active site. Sometimes, the reductase and the ferredoxin are fused in a single polypeptide. The oxygenase component is a multimeric protein, with either an n n (n=2 or 3) or 3 structure, that contains one [2Fe-2S] Rieske cluster and one non-heme iron atom per subunit (1). During a catalytic cycle, two electrons from the reduced pyridine nucleotide are transferred, via the reductase, the ferredoxin and the Rieske center, to the Fe(II) ion at the active site. The reducing equivalents allow the activation of molecular oxygen, which is a prerequisite to dihydroxylation of the substrate (10). So far, only a few RHDs have been purified and extensively characterized, including phthalate dioxygenase (11, 12), naphthalene dioxygenase (13, 14) and biphenyl dioxygenase (15). None of these enzymes is able to oxidize substrates with more than three fused rings, and data on the mechanism, kinetics and efficiency of the oxidation of high molecular weight PAHs by bacterial dioxygenases are relatively scarce (16). However, the four-ring PAHs chrysene and benz[a]anthracene, and the five-ring benzo[a]pyrene are of particular concern because they are well-documented carcinogens (17). Recently, a Sphingomonad endowed
A Putative Monooxygenase Mimic Which Functions via Well-Disguised Free Radical Chemistry1
Journal of the American Chemical Society, 1997
The hydroxylation of cycloalkanes at 25°C by the syringe pump addition of tert-alkyl hydroperoxides (10 and 1 equiv based on catalyst) to deoxygenated acetonitrile containing cycloalkanes (0.64 M) and 0.61 mM of the catalyst, [Fe III 2 O(TPA) 2 (H 2 O) 2 ] 4+ , is demonstrated to be a reaction which involves freely diffusing cycloalkyl radicals, i.e., free alkyl radicals. In recent years there have been many attempts to mimic the chemistry of monooxygenases such as cytochrome P450 and methane monooxygenase which can oxidize saturated hydrocarbons by processes which do not involve free (i.e., freely diffusing) radicals. 3 Following the lead provided by these enzymes, (ferric) iron has generally been chosen as the catalytically active metal and two-electron-reduced oxygen, in the form of H 2 O 2 or tert-butyl hydroperoxide (TBHP), has been utilized (to avoid the requirement for a sacrificial reductant). However, Fe III /TBHP systems may not undergo the desired heterolysis to give a high-valent iron-oxo species (formally Fe V dO) as is believed to occur when monooxygenases react with hydroperoxides. Instead, a homolysis may occur to form free tert-butoxyl radicals which then dominate the subsequent chemistry (see Scheme 1). The oxidation of an alkane to a mixture of alcohol, ketone, and the mixed peroxide (shown in bold face in the scheme) is a very clear indication that free-radical chemistry has occurred. Unfortunately, this signature has all too frequently been ignored. 2-Methyl-1-phenyl-2-propyl hydroperoxide (MPPH) is a probe capable of distinguishing between free alkoxyl radical chemistry and radical-free (enzyme mimetic) chemistry in iron/ tert-alkyl hydroperoxide/hydrocarbon oxidation systems. 4 This probe relies on the fact that if the corresponding tert-alkoxyl radical were formed and diffused from its site of formation into the bulk solution it would undergo far too rapid a-scission (k ∼ 2 × 10 8 s-1) for it to abstract a hydrogen atom from a saturated hydrocarbon, i.e., the equivalent of reaction 8 cannot occur. MPPH has been employed at the NRC in Ottawa to demonstrate that cycloalkane oxidations using TBHP and two tris(2-pyridinylmethyl)amine (TPA) complexes, 5 [Fe III Cl 2-(TPA)] + and [Fe III 2 O(OAc)(TPA) 2 ] 3+ , and a Fe III picolinate/ pyridine complex 6 all occurred via straighforward free radical † SIMS, NRC.
Journal of Biological Chemistry, 2000
Naphthalene 1,2-dioxygenase (NDOS) is a three-component enzyme that catalyzes cis-(1R,2S)-dihydroxy-1,2dihydronaphthalene formation from naphthalene, O 2 , and NADH. We have determined the conditions for a single turnover of NDOS for the first time and studied the regulation of catalysis. As isolated, the ␣ 3  3 oxygenase component (NDO) has up to three catalytic pairs of metal centers (one mononuclear Fe 2؉ and one diferric Rieske iron-sulfur cluster). This form of NDO is unreactive with O 2 . However, upon reduction of the Rieske cluster and exposure to naphthalene and O 2 , ϳ0.85 cisdiol product per occupied mononuclear iron site rapidly forms. Substrate binding is required for oxygen reactivity. Stopped-flow and chemical quench analyses indicate that the rate constant of the single turnover product-forming reaction significantly exceeds the NDOS turnover number. UV-visible and electron paramagnetic resonance spectroscopies show that during catalysis, one mononuclear iron and one Rieske cluster are oxidized per product formed, satisfying the two-electron reaction stoichiometry. The addition of oxidized or reduced NDOS ferredoxin component (NDF) increases both the product yield and rate of oxidation of formerly unreactive Rieske clusters. The results show that NDO alone catalyzes dioxygenase chemistry, whereas NDF appears to serve only an electron transport role, in this case redistributing electrons to competent active sites.
European Journal of Biochemistry, 1997
Nocurdiu corultina B-276 possesses a constitutive multi-component alkene monooxygenase which catalyses the epoxidation of terminal and sub-terminal alkenes. The epoxygenase component of this system has been purified with an overall yield of 35%. The electron paramagnetic resonance spectrum of the oxidised protein has a weak signal at g = 4.3, which we ascribe to rhombic iron and a free radical signal at g,,, = 2.01. Upon partial reduction with dithionite using methyl viologen as a mediator, a signal at g,,, = 1.9 appeared. Upon further reduction with excess dithionite a signal at g = 15 appeared with the concomitant disappearance of the g,,, = 1.9 signal. These results indicate that the epoxygenase contains a bridged dinuclear iron centre similar to that found in a variety of proteins involved in oxygen transport and activation as well as desaturation of fatty acids. Analysis of the products of the reaction indicates that A M 0 is capable of stereospecific epoxidation of alkenes producing the R-enantiomer in high yield, a reaction catalysed by very few oxygenase enzymes. Whole cells gave lower enantiomeric excess values for the epoxide and a stereospecific epoxidase enzyme has been proposed to account for this difference. Although alkene monooxygenase was not inhibited by ethyne, a potent inhibitor of soluble methane monooxygenase with which alkene monooxygenase shares many common features, it was weakly inhibited by propyne with an apparent K, value of 340 pM. The mechanistic implications of these physicochemical features of the enzyme are discussed.
Biochemistry, 2004
1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) is a cofactor-less dioxygenase belonging to the R/ hydrolase fold family, catalyzing the cleavage of 1H-3-hydroxy-4-oxoquinaldine (I) and 1H-3-hydroxy-4-oxoquinoline (II) to N-acetyl-and N-formylanthranilate, respectively, and carbon monoxide. Bisubstrate steady-state kinetics and product inhibition patterns of HodC, the C69A protein variant of Hod, suggested a compulsory-order ternary-complex mechanism, in which binding of the organic substrate precedes dioxygen binding, and carbon monoxide is released first. The specificity constants, k cat /K m,A and k cat /K m,O 2 , were 1.4 × 10 8 and 3.0 × 10 5 M -1 s -1 with I and 1.2 × 10 5 and 0.41 × 10 5 M -1 s -1 with II, respectively. Whereas HodC catalyzes formation of the dianion of its organic substrate prior to dioxygen binding, HodC-H251A does not, suggesting that H251, which aligns with the histidine of the catalytic triad of the R/ hydrolases, acts as general base in catalysis. Investigation of base-catalyzed dioxygenolysis of I by electron paramagnetic resonance (EPR) spectroscopy revealed formation of a resonance-stabilized radical upon exposure to dioxygen. Since in D 2 O spectral properties are not affected, exchangeable protons are not involved, confirming that the dianion is the reactive intermediate that undergoes single-electron oxidation. We suggest that in the ternary complex of the enzyme, direct single-electron transfer from the substrate dianion to dioxygen may occur, resulting in a radical pair. Based on the estimated spin distribution within the radical anion (observed in the model reaction of I), radical recombination may produce a C4or C2-hydroperoxy(di)anion. Subsequent intramolecular attack would result in the 2,4-endoperoxy (di)anion that may collapse to the reaction products. † Supported by the Deutsche Forschungsgemeinschaft (Grants FE 383/5-1 and FE 383/5-2).