Intradiol Cleavage Research Articles

Nuclear Resonance Vibrational Spectroscopy Definition of Peroxy Intermediates in Catechol Dioxygenases: Factors that Determine Extra- versus Intradiol Cleavage.

The extradiol dioxygenases (EDOs) and intradiol dioxygenases (IDOs) are nonheme iron enzymes that catalyze the oxidative aromatic ring cleavage of catechol substrates, playing an essential role in the carbon cycle. The EDOs and IDOs utilize very different FeII and FeIII active sites to catalyze the regiospecificity in their catechol ring cleavage products. The factors governing this difference in cleavage have remained undefined. The EDO homoprotocatechuate 2,3-dioxygenase (HPCD) and IDO protocatechuate 3,4-dioxygenase (PCD) provide an opportunity to understand this selectivity, as key O2 intermediates have been trapped for both enzymes. Nuclear resonance vibrational spectroscopy (in conjunction with density functional theory calculations) is used to define the geometric and electronic structures of these intermediates as FeII-alkylhydroperoxo (HPCD) and FeIII-alkylperoxo (PCD) species. Critically, in both intermediates, the initial peroxo bond orientation is directed toward extradiol product formation. Reaction coordinate calculations were thus performed to evaluate both the extra- and intradiol O-O cleavage for the simple organic alkylhydroperoxo and for the FeII and FeIII metal catalyzed reactions. These results show the FeII-alkylhydroperoxo (EDO) intermediate undergoes facile extradiol O-O bond homolysis due to its extra e-, while for the FeIII-alkylperoxo (IDO) intermediate the extradiol cleavage involves a large barrier and would yield the incorrect extradiol product. This prompted our evaluation of a viable mechanism to rearrange the FeIII-alkylperoxo IDO intermediate for intradiol cleavage, revealing a key role in the rebinding of the displaced Tyr447 ligand in this rearrangement, driven by the proton delivery necessary for O-O bond cleavage.

Iron(III) complexes of pyrrolidine and piperidine appended tridentate 3N donor ligands as models for catechol dioxygenase enzymes

Six new mononuclear iron(III) complexes [Fe(L1)Cl3]-[Fe(L6)Cl3] bearing tridentate all-nitrogen donor ligands (where L1 = N-(pyridin-2-ylmethyl)-2-(pyrrolidin-1-yl)ethanamine, L2 = 2-(piperidin-1-yl)-N-(pyridin-2-ylmethyl)ethanamine, L3 = 1-(piperidin-2-yl)-N-(pyridin-2-ylmethyl)methanamine, L4 = N-((1-methyl-1H-imidazol-2-yl)methyl)-2-(pyrrolidin-1-yl)ethan-1-amine, L5 = N-((1-methyl-1H-imidazol-2-yl)methyl)-2-(piperidin-1-yl)ethan-1-amine), L6 = 1-(1-methyl-1H-imidazol-2-yl)-N-(piperidin-2-ylmethyl)methanamine) have been prepared, characterized and evaluated as the functional models for the carbon–carbon bond-cleaving catechol dioxygenase enzymes. The molecular structure of complexes 1 and 3 revealed a distorted octahedral geometry around iron(III) center with facial coordination of ligands via three nitrogen atoms and three chloride ions occupy the remaining sites of the octahedron. Iron(III) catecholate adducts were generated in situ by adding one equivalent of various catechols pretreated with two equivalent of triethylamine. The 3,5-di-tert-butylcatecholate adduct of the complexes 1–6 show two intense bands in visible region (540–900 nm) as the result of DBC2--to-iron(III) ligand-to-metal charge-transfer transitions in acetonitrile. During electrochemical analysis, all the complexes display FeIII→FeII redox couple in the similar potential range (−0.260 to −0.336 V). The dioxygenase activity of present complexes produces major amount of extradiol cleavage products (75–92%) over the intradiol cleavage products (4–14%) at moderate rate (0.561–1.744 × 10−2 M−1 s−1). Interestingly, the iron(III) complexes 3 and 6 with facially coordinating tridentate ligands with heterocyclic piperidine amine (NH) nitrogen donor moiety produce major amounts of extradiol-cleavage products (3, 89%; 6, 75%) along with only minor amounts of other product (10–24%) without any intradiol-cleavage products.

Synthesis, characterization and O2 reactivity of a bioinspired cobalt(II)-catecholate complex

Inspired by the active site structure of Co-HPCD (HPCD = homoprotocatechuate 2,3-dioxygenase), in which the catechol binds to the cobalt(II) center in its monoanionic form in a bidentate fashion, we report the synthesis and characterization of [PhTttBu]Co(3,5-DBCatH) [PhTttBu = phenyltris(tertbutylthiomethyl)borate; 3,5-DBCatH = 3,5-di-tert-butyl-catecholate monoanion], the first example of a well-characterized mononuclear cobalt(II) complex of a monoanionic catecholate ligand. The net reaction of hydrogen atom transfer (HAT) occurs immediately when [PhTttBu]Co(3,5-DBCatH) is exposed to O2, generating a previously reported five-coordinate Co(II)-semiquinonate complex, [PhTttBu]Co(3,5-DBSQ) (3,5-DBSQ = 3,5-di-tert-butyl-1,2-semiquinonate). At low temperature, O2 uptake by [PhTttBu]Co(3,5-DBSQ) was observed by electronic absorption spectroscopy. Upon warming to room temperature, [PhTttBu]Co(3,5-DBSQ) decays slowly under an O2 atmosphere releasing muconic anhydride, an intradiol cleavage product, in 16% yield as previously reported. Kinetic measurements reveal that the decay of [PhTttBu]Co(3,5-DBSQ) under excess O2 exhibits pseudo-first-order behavior. Mechanistic considerations involving Co(III)-superoxo and Co(III)-alkylperoxo intermediates are presented based on the above-mentioned data and the metal ion effect on the O2 reactivity.

Catalytic Fate of Two Copper Complexes towards Phenoxazinone Synthase and Catechol Dioxygenase Activity

AbstractIn this present work, we report the synthesis and structural characterization of two copper(II) complexes, [Cu(bpy)Cl2] (1) & [Cu(μ‐Cl)(phen)Cl]2 (2) [bpy=2,2′‐bipyridine; phen=1,10‐phenanthroline]. We have also studied their catalytic fate towards phenoxazinone synthase and catechol dioxygenase activity. X‐ray structural analyses revealed that 1 & 2 crystallize in triclinic & monoclinic system with P 1 and Cc space group respectively. The copper complexes catalyse the oxidative coupling of 2‐amino phenol (2‐AP) to aminophenoxazin‐3‐one with significant turn over number, kcat(h−1)=2.08×103 & 2.16×103 for 1 & 2 resectively. During investigation of catechol dioxygenase activity, stoichiometric addition of 1 & 2 to 3,5‐di‐tert‐butylcatechol (DTBC) in acetonitrile produce in situ catecholate‐to‐Cu(II) absorption bands at 812 and 821 nm respectively. The in situ Cu(II)‐catecholate species for both 1 & 2 react with molecular oxygen at the rate, kobs: 7.95×10−4 and 1.30×10−3 min−1 respectively and produce intradiol cleavage products in exclusive amount. Minor amount of benzoquinone is also found in solution. Intradiol products are found as major product in solution and accounts in favour of substrate activation mechanism.

Catalytic aspects of a copper(II) complex: biological oxidase to oxygenase activity

A coper(II) complex, $$\hbox {[Cu(dpa)}_{2}\hbox {(OAc)](ClO}_{4})$$ (1) [dpa = $$ 2,2^\prime $$ -dipyridylamine; OAc $$=$$ acetate], has been synthesized and crystallographically characterized. X-ray structure analysis revealed that this mononuclear Cu(II) complex crystallizes as a rare class of hexa coordination geometry named bicapped square pyramidal geometry with $$P2_{1}/\hbox {c}$$ space group. This copper complex displays excellent catalytic efficiency, $$\hbox {k}_{\mathrm{cat}}/\hbox {K}_{\mathrm{M} }(\hbox {h}^{-1}) = 6.17\times 10^{5}$$ towards the oxidative coupling of 2-aminophenol (2-AP) to aminophenoxazin-3-one. Further, upon stoichiometric addition of copper(II) complex to 3,5-DTBC in presence of molecular oxygen in ethanol medium, the copper complex affords predominantly extradiol cleavage products along with a small amount of benzoquinone and a trace amount of intradiol cleavage products at a rate, $$\hbox {k}_{\mathrm{obs}}= 1.09\times 10^{-3}$$ $$\hbox {min}^{-1}$$ , which provide substantial evidence for the oxygen activation mechanism. This paper presents a novel addition of a copper(II) complex having the potential to mimic the active site of phenoxazinone synthase and catechol dioxygenase enzymes with significant catalytic efficiency. SYNOPSIS The mononuclear copper complex having unusual hexa coordination geometry exhibits significant catalytic efficiency, $$\hbox {k}_{\mathrm{cat}}/\hbox {K}_{\mathrm{M}}(\hbox {h}^{-1}) = 6.17\times 10^{5}$$ towards oxidation of 2-aminophenol which predominantly produced extradiol cleavage products at a rate, $$\hbox {k}_{\mathrm{obs}}= 1.09\times 10^{-3}$$ $$\hbox {min}^{-1}$$ upon addition of 3,5-DTBC in presence of molecular oxygen.

Electronic, Magnetic, and Redox Properties and O2 Reactivity of Iron(II) and Nickel(II) o-Semiquinonate Complexes of a Tris(thioether) Ligand: Uncovering the Intradiol Cleaving Reactivity of an Iron(II) o-Semiquinonate Complex.

The iron(II) semiquinonate character within the iron(III) catecholate species has been proposed by numerous studies to account for the O2 reactivity of intradiol catechol dioxygenases, but a well-characterized iron(II) semiquinonate species that exhibits intradiol cleaving reactivity has not yet been reported. In this study, a detailed electronic structure description of the first iron(II) o-semiquinonate complex, [PhTttBu]Fe(phenSQ) [PhTttBu = phenyltris(tert-butylthiomethyl)borate; phenSQ = 9,10-phenanthrenesemiquinonate; Wang et al. Chem. Commun. 2014, 50, 5871-5873], was generated through a combination of electronic and Mössbauer spectroscopies, SQUID magnetometry, and density functional theory (DFT) calculations. [PhTttBu]Fe(phenSQ) reacts with O2 to generate an intradiol cleavage product, diphenic anhydride, in 16% yield. To assess the dependence of the intradiol reactivity on the identity of the metal ion, the nickel analogue, [PhTttBu]Ni(phenSQ), and its derivative, [PhTttBu]Ni(3,5-DBSQ) (3,5-DBSQ = 3,5-di-tert-butyl-1,2-semiquinonate), were prepared and characterized by X-ray crystallography, mass spectrometry, 1H NMR and electronic spectroscopies, and SQUID magnetometry. DFT calculations, evaluated on the basis of the experimental data, support the electronic structure descriptions of [PhTttBu]Ni(phenSQ) and [PhTttBu]Ni(3,5-DBSQ) as high-spin nickel(II) complexes with antiferromagnetically coupled semiquinonate ligands. Unlike its iron counterpart, [PhTttBu]Ni(phenSQ) decomposes slowly in an O2 atmosphere to generate 14% phenanthrenequinone with a negligible amount of diphenic anhydride. [PhTttBu]Ni(3,5-DBSQ) does not react with O2. This dramatic effect of the metal-ion identity supports the hypothesis that a metal(III) alkylperoxo species serves as an intermediate in the intradiol cleaving reactions. The redox properties of all three complexes were probed using cyclic voltammetry and differential pulse voltammetry, which indicate an inner-sphere electron-transfer mechanism for the formation of phenanthrenequinone. The lack of O2 reactivity of [PhTttBu]Ni(3,5-DBSQ) can be rationalized by the high redox potential of the metal-ligated 3,5-DBSQ/3,5-DBQ couple.

Catalytic aspects of a nickel(II)–bipyridine complex towards phosphatase and catechol dioxygenase activity

A mononuclear nickel(II) complex, [Ni(bpy)2(OH2)(NO3)](NO3) (1) [bpy=2,2′-bipyridine], has been synthesised and structurally characterised by routine analytical techniques including a single crystal X-ray diffraction study. X-ray crystal structure analysis shows that 1 crystallises in the triclinic system with the P1¯ space group and adopts an octahedral geometry. The nickel(II) complex has been evaluated as a functional model for phosphatase and catechol dioxygenase enzymes by using 4-nitrophenylphosphate (PNPP) and 3,5-di-tert-butylcatechol (DTBC) as model substrates in aqueous-DMF and acetonitrile media respectively. This mononuclear nickel complex exhibits good hydrolytic phosphoester cleavage efficiency, viz. a kcat value of 8.42×103h−1, and produces mostly benzoquinone (kobs=2.3×10−3min−1) and intradiol cleavage products during an investigation of catechol dioxygenase activity.

Catalytic promiscuity of an iron(II)–phenanthroline complex

A mononuclear iron(II) complex, [Fe(phen)3]Cl2 (1) (phen =1,10‐phenanthroline), has been synthesized in crystalline phase and characterized using various spectroscopic techniques including single crystal X‐ray diffraction. Crystal structure analysis revealed that 1 crystallizes in a monoclinic system with C2/m space group. Complex 1 acts as a functional model for a biomimetic catalyst promoting the aerobic oxidation of 3,5‐di‐tert‐butylcatechol (3,5‐DTBC) through radical pathways with a significant turnover number (kcat =3.55 × 103 h−1) and exhibits catechol dioxygenase activity towards the same 3,5‐DTBC substrate at room temperature in oxygen‐saturated ethanol medium. The existence of an isobestic point at 610 nm from spectrophotometric data indicates the presence of Fe3+ −3,5‐DTBC adduct favouring an enzyme–substrate binding phenomenon. Upon stoichiometric addition of 3,5‐DTBC pretreated with two equivalents of triethylamine to the iron complex, two catecholate‐to‐iron(III) ligand‐to‐metal charge transfer bands (575 and 721 nm) are observed and the in situ generated catecholate intermediate reacts with dioxygen (kobs =9.89 × 10−4 min−1) in ethanol medium to afford exclusively intradiol cleavage products along with a small amount of benzoquinone, and a small amount of extradiol cleavage products, which provide substantial evidence for a substrate activation mechanism. Copyright © 2016 John Wiley & Sons, Ltd.

Unravelling the Molecular Origin of the Regiospecificity in Extradiol Catechol Dioxygenases.

Many factors have been suggested to control the selectivity for extradiol or intradiol cleavage in catechol dioxygenases. The varied selectivity of model complexes and the ability to force an extradiol enzyme to do intradiol cleavage indicate that the problem may be complex. In this paper we focus on the regiospecificity of the proximal extradiol dioxygenase, homoprotocatechuate 2,3-dioxygenase (HPCD), for which considerable advances have been made in our understanding of the mechanism from an experimental and computational standpoint. Two key steps in the reaction mechanism were investigated: (1) attack of the substrate by the superoxide moiety and (2) attack of the substrate by the oxyl radical generated by O-O bond cleavage. The selectivity at both steps was investigated through a systematic study of the role of the substrate and the first and second coordination spheres. For the isolated native substrate, intradiol cleavage is calculated to be both kinetically and thermodynamically favored, therefore nature must use the enzyme environment to reverse this preference. Two second sphere residues were found to play key roles in controlling the regiospecificity of the reaction: Tyr257 and His200. Tyr257 controls the selectivity by modulating the electronic structure of the substrate, while His200 controls selectivity through steric effects and by preventing alternative pathways to intradiol cleavage.

Oxidant dependent oxidation of copper bound catecholate: Catecholase versus catechol dioxygenase activity

A series of new mixed ligand copper complexes CuII(PBI)(O2Ncat) (1) (PBI=2-(2-pyridyl)-benzimidazole), CuII(DMPBI)(O2Ncat) (2) (DMPBI=5,6-dimethyl-2-(2-pyridyl)-benzimidazole), CuII(PBO)(O2Ncat) (3) (PBO=2-(2-pyridyl)-benzoxazole), CuII(PBT)(O2Ncat) (4) (PBT=2-(2-pyridyl)-benzothiazole), and Cu(TBA)(O2Ncat) (5) (TBA=4-(1H-1,3-benzodiazol-2-yl)-1,3-thiazole) have been synthesized by classic organic, bioinorganic methods. The complexes have been found to mimic catechol oxidase and catechol dioxygenase activity depending on the oxidant used. Triplet state dioxygen activates the complex leading to intradiol cleavage product, 2-nitromuconic acid while sterically more demanding tert-butyl hydroperoxide (TBHP) oxidizes the 4-nitrocatechol (4-O2NcatH2) ligand into 4-nitrobenzoquinone. We have also found that the electron density on the copper center significantly increase the reactivity toward the oxidant used.

Bioinspired copper(I) complexes that exhibit monooxygenase and catechol dioxygenase activity.

New tripodal ligand L2 featuring three different pyridyl/imidazolyl-based N-donor units at a bridgehead C atom, from which one of the imidazolyl units is separated by a phenylene linker, was synthesized and investigated with regards to copper(I) complexation. The resulting complex [(L2)Cu]OTf (2(OTf)), the known complex [(L1)Cu]OTf (1(OTf); L1 differs from L2 in that it lacks the phenylene spacer) and [(L3)Cu]OTf (3(OTf)), prepared from a known chiral, tripodal, N-donor ligand featuring pyridyl, pyrazolyl, and imidazolyl donors, were tested as catalysts for the oxidation of sodium 2,4-di-tert-butylphenolate (NaDTBP) with O2. Indeed, they mediated NaDTBP oxidation to give mainly the corresponding catecholate and quinone (Q). None of the complexes 1(OTf), 2(OTf), and 3(OTf) is superior to the others, as yields were comparable and, if the presence of protons is guaranteed by concomitant addition of the phenol DTBP, the oxidation can also be performed catalytically. For all complexes stoichiometric oxidations under certain conditions (concentrated solutions, high NaDTBP content) were found to also generate products typical for metal-mediated intradiol cleavage of the catecholate with O2. As shown representatively for 1(OTf) this dioxygenation sets in at a later stage of the reaction. Initially a copper species responsible for the monooxygenation must form from 1(OTf)/NaDTBP/O2, and only thereafter is the copper species responsible for dioxygenation formed and consumes Q as substrate. Hence, under these circumstances complexes 1(OTf)-3(OTf) show both monooxygenase and catechol dioxygenase activity.

Biomimetic iron(iii) complexes of facially and meridionally coordinating tridentate 3N ligands: tuning of regioselective extradiol dioxygenase activity in organized assemblies

Four mononuclear iron(III) complexes of the type [Fe(L)Cl3] 1-4, where L is a tridentate 3N ligand such as (2-pyridin-2-ylethyl)(pyridin-2-ylmethyl)amine (L1), (methyl)(2-pyridin-2-ylethyl)(pyridin-2-ylmethyl)amine (L2), bis(pyridin-2-ylethyl)amine (L3), and (1-methyl-1H-imidazol-2-ylmethyl)(pyridin-2-ylethyl)amine (L4), have been isolated and studied as functional models for catechol dioxygenase enzymes. In [Fe(L2)Cl3] 2, the ligand L2 is coordinated facially to iron(III) whereas in [Fe(L1)Cl3] 1 and [Fe(L4)Cl3] 4 the ligands L1 and L4 are coordinated meridionally. In DCM, CH3CN and aqueous SDS, CTAB and TX-100 micellar media, the positions of both the low and high energy catecholate-to-iron(III) LMCT bands (465-530, 690-860 nm) observed for the 3,4-di-tert-butylcatecholate (DBC(2-)) adducts of the iron(III) complexes vary in the order 2 > 1 > 3 > 4, which reflects the influence of the stereoelectronic factors, mode of coordination and the chelate ring size formed by the tridentate ligands. Spectral and electrochemical studies disclose the formation and location of the cationic adducts as solvated [Fe(L)(DBC)(H2O)](+) species mostly in the aqueous micellar pseudophases of SDS and TX-100 and in the aqueous phase of CTAB micellar solution. The [Fe(L)(DBC)Cl] adducts of 1, 3 and 4, generated in situ, afford major amounts of intradiol cleavage products (17.0-70.0%) and smaller amounts of extradiol (1.2-4.2%) products with varying extradiol to intradiol cleavage product selectivity (E/I: 1, 0.08 : 1; 3, 0.02 : 1; 4, 0.3 : 1). On the other hand, interestingly, the adduct [Fe(L2)(DBC)Cl] of 2 generated in DCM yields a major amount of extradiol (54.0%) and a lower amount (18.3%) of the intradiol cleavage products (E/I, 3 : 1). Remarkably, in aqueous SDS micellar media, it shows exclusive extradiol cleavage products (79.4%) while all the other complexes show very low selectivity (E/I: 1, 0.03 : 1; 2, 79.4 : 0, 3, 0.06 : 1, 4, 0.06 : 1), suggesting the suitability of SDS medium for 2 to elicit exclusive extradiol cleavage. The TX-100 micellar medium also provides a suitable hydrophobic environment for 2 to elicit extradiol cleavage. However, in CTAB micellar medium, 2 shows cleavage selectivity lower than others. Also, the rate of dioxygenation is higher in SDS micellar medium than in DCM, and is dependent upon the chelate ring size.

Fusion of Dioxygenase and Lignin-binding Domains in a Novel Secreted Enzyme from Cellulolytic Streptomyces sp. SirexAA-E

Streptomyces sp. SirexAA-E is a highly cellulolytic bacterium isolated from an insect/microbe symbiotic community. When grown on lignin-containing biomass, it secretes SACTE_2871, an aromatic ring dioxygenase domain fused to a family 5/12 carbohydrate-binding module (CBM 5/12). Here we present structural and catalytic studies of this novel fusion enzyme, thus providing insight into its function. The dioxygenase domain has the core β-sandwich fold typical of this enzyme family but lacks a dimerization domain observed in other intradiol dioxygenases. Consequently, the x-ray structure shows that the enzyme is monomeric and the Fe(III)-containing active site is exposed to solvent in a shallow depression on a planar surface. Purified SACTE_2871 catalyzes the O2-dependent intradiol cleavage of catechyl compounds from lignin biosynthetic pathways, but not their methylated derivatives. Binding studies show that SACTE_2871 binds synthetic lignin polymers and chitin through the interactions of the CBM 5/12 domain, representing a new binding specificity for this fold-family. Based on its unique structural features and functional properties, we propose that SACTE_2871 contributes to the invasive nature of the insect/microbial community by destroying precursors needed by the plant for de novo lignin biosynthesis as part of its natural wounding response.

Kβ Detected High‐Resolution XANES of FeII and FeIII Models of the 2‐His‐1‐Carboxylate Motif: Analysis of the Carboxylate Binding Mode

AbstractProteins sharing the same “2‐His‐1‐carboxylate” structural motif have little amino acid sequence similarity and are able to perform many different reactions. Many factors have been cited to explain their different specificity and turnover rates, like protein environment, coordinated ligand geometry, electronic structure of the active site, etc. In this paper, we present a combined approach applying high‐resolution XANES spectroscopy and theory simulations to different model complexes that mimic the binding modes of the amino acids to the metal site. Experiments were performed on three compounds showing three metal sites: ferrous hexacoordinate, ferric pentacoordinate and ferrous pentacoordinate. The first two compounds bear an N,N,O‐tridentate 3,3‐bis(1‐alkylimidazol‐2‐yl)propionate ligand that features a monodentate carboxylate group. These complexes mimic the activity of extradiol dioxygenases but also exhibit intradiol cleavage activity. The third compound features a bidentate terphenylcarboxylate ligand and a sterically hindered bidentate N,N‐donor, thus providing a good structural mimic of the ternary enzyme‐tetrahydrobiopterin‐substrate complex in pterin‐dependent phenylalanine hydroxylase, which also contains a bidentate carboxylate. Modeling of high‐resolution XANES on well‐defined model complexes of different geometry can aid in protein structure elucidation. XANES gives the oxidation state and coordination number of the metal in the non‐crystallized protein at natural pH. The accuracy of the results is limited by the core‐hole and experimental broadenings. We found that high‐resolution XANES experiments give increased resolution at the pre‐edges, but limited improvement at the main edge. These high‐resolution pre‐edges can be accurately simulated by using crystal field multiplet theory (CFM). We show that by combining modelling and XANES simulations with FEFF8, detailed structural and chemical information can be obtained. We found that a short O2–metal distance for the carboxylate oxygen atom not bound to the metal causes a higher white line in FeII, which is similar to the results obtained for the pterin‐dependent hydroxylase, tyrosine hydroxylase (TYH). Full‐potential FDMNES simulations for each sample confirm the accuracy of the main results with muffin‐tin approximation (FEFF8).

Oxidation versus Dioxygenation of Catechol: The Iron–Bispidine System

AbstractThe iron–bispidine‐catalyzed oxidation and dioxygenation (catechol dioxygenase activity) [i.e., the oxidation of 3,5‐di‐tert‐butylcatecholate (dbc2–) by [FeII(L)X2]n+ (L = 3,7‐dimethyl‐9‐oxo‐2,4‐bis(2‐pyridyl)‐3,7‐diazabicyclo[3.3.1]nonane‐1,5‐dicarboxylate methyl ester)] and air (O2) was studied experimentally and supported by the analysis of the X‐ray crystal structure of [Fe(L·MeOH)(tcc)][B(Ph)4] with the deactivated tetrachlorocatecholate tcc2– and a DFT‐based analysis. The [FeII(L)X2]n+/O2/dbc2– system catalyzes the intradiol cleavage of dbc2– but with a relatively low activity (5 % yield); most of the substrate is oxidized in a two‐electron oxidation to the benzoquinone (dbq) product (48 % yield). The crystallographic and DFT‐based theoretical analyses indicate that this is due to the high oxidation potential of the FeIII oxidant (fast and efficient electron transfer), that is, the oxidation to the benzoquinone side product is faster, and due to the bonding mode of the catecholate substrate to the FeIII oxidant, with little spin density transferred to the catecholate substrate.

Functional models for enzyme–substrate adducts of catechol dioxygenase enzymes: The Lewis basicity of facially coordinating tridentate phenolate ligands tunes the rate of dioxygenation and product selectivity

A few iron(III) 3,5-di- tert-butylcatecholate (DBC 2−) adducts of the type [Fe(L)(DBC)(CH 3OH)], where L is a tridentate substituted monophenolate ligand such as 2-(( N-benzylpyrid-2-ylmethylamino)methyl)phenol (H(L1)), 2-(( N-benzylpyrid-2-ylmethylamino)-methyl)-4,6-dimethylphenol (H(L2)), 2-(( N-benzylpyrid-2-ylmethylamino)methyl)-4,6-di- tert-butylphenol (H(L3)) and 2-(( N-benzylpyrid-2-ylmethylamino)methyl)-4-nitrophenol (H(L4)), have been isolated and characterized by elemental and ESI-MS analysis. The spectral and electrochemical properties and dioxygenase activities of the adducts have been studied in methanol solution. Upon varying the substituents on the phenolate ring from electron-releasing to electron-withdrawing, the redox potential of DBSQ/DBC 2− couple is shifted to a more positive value indicating an increase in covalency of iron(III)–catecholate bonds. All the complexes elicit cleavage of DBC 2− using molecular oxygen to afford both intra- (I) and extradiol (E) cleavage products with the product selectivity (E/I) varying in the range 0.3–1.9. Interestingly, the incorporation of electron-withdrawing substituents facilitates the regioselective extradiol cleavage of catechol while that of electron-releasing substituents facilitate the regioselective intradiol cleavage.

Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. DS002 and Cloning, Sequencing of Partial catA Gene

Catechol 1,2-dioxygenase (C12O) was purified to electrophoretic homogeneity from Acinetobacter sp. DS002. The pure enzyme appears to be a homodimer with a molecular mass of 66kDa. The apparent K(m) and V(max) for intradiol cleavage of catechol were 1.58μM and 2 units per mg of protein respectively. Unlike other C12Os reported in the literature, the catechol 1,2-dioxygenase of Acinetobacter showed neither intradiol nor extradiol cleavage activity when substituted catechols were used as substrates. However, it has shown mild intradiol cleavage activity when benzenetriol was used as substrate. As determined by two dimensional electrophoresis (2DE) followed MALDI-TOF/TOF analyses and gel permeation chromatography, no isoforms of C12O was observed in Acinetobacter sp. DS002. Further, the C12O was seen only in cultures grown in benzoate and it was completely absent in succinate grown cultures. Based on the sequence information obtained from MS/MS data, degenerate primers were designed to amplify catA gene from the genomic DNA of Acinetobacter sp. DS002. The sequence of the PCR amplicon and deduced amino acid sequence showed 97% similarity with a catA gene of Acinetobacter baumannii AYE (YP_001713609).

Theoretical Insights into Heme-Catalyzed Oxidation of Cyclohexane to Adipic Acid

Adipic acid is a key compound in the chemical industry, where it is mainly used in the production of polymers. The conventional process of its generation requires vast amounts of energy and, moreover, produces environmentally deleterious substances. Thus, there is interest in alternative ways to gain adequate amounts of adipic acid. Experimental reports on a one-pot iron-catalyzed conversion of cyclohexane to adipic acid motivated a theoretical investigation based on density functional theory calculations. The process investigated is interesting because it requires less energy than contemporary methods and does not produce environmentally harmful side products. The aim of the present contribution is to gain insight into the mechanism of the iron-catalyzed cyclohexane conversion to provide a basis for the further development of this process. The rate-limiting step of the process is discussed, but considering the accuracy of the calculations, it is difficult to ensure whether the rate-limiting step is in the substrate oxidation or in the generation of the catalytically active species. It is shown that the slowest step in the substrate oxidation is the conversion of cyclohexanol to cyclohexane-1,2-diol. Hydrogen-atom transfer from one of the OH groups of cyclohexane-1,2-diol makes the intradiol cleavage occur spontaneously.

Mononuclear iron(iii) complexes of 3N ligands in organized assemblies: spectral and redox properties and attainment of regioselective extradiol dioxygenase activity

Four octahedral iron(III) complexes of the type [Fe(L)Cl(3)], where L is a tridentate 3N ligand like N,N-bis(pyrid-2-ylmethyl)amine (bpa, L1), N,N-bis(benzimidazol-2-ylmethyl)amine (bba, L2), 1,4,7-triazacyclononane (tacn, L3) and 2,2';6',2''-terpyridine (terpy, L4), have been isolated and their catechol dioxygenase activity investigated in dichloromethane, water and different aqueous micellar media. The positions of both the catecholato-to-iron(III) LMCT bands observed for the DBC(2-) (H(2)DBC = 3,5-di-tert-butylcatechol) adducts reveal that the adducts are present as cationic [Fe(L)(DBC)(H(2)O)](+) species, which interact strongly with anionic SDS micelles and dock themselves on the anionic micellar surface, and that they exist in the aqueous phase in CTAB and TX 100 micelles. The Fe(III)/Fe(II) redox potentials of the complexes throw light on the Lewis acidity of the iron(III) center as modified by the ligand donor atoms and hence the interaction of the complexes with different micelles. The DBSQ/DBC(2-) redox potentials in SDS micellar media are more positive than those in aqueous solution confirming the presence of the aqua species [Fe(L)(DBC)(H(2)O)](+). The DBC(2-) adducts of the iron(III) complexes of bpa, bba and tacn ligands, all with facial coordination, elicit extradiol (E) cleavage to different extents while the adduct of the terpy complex with meridional coordination of the ligand shows always intradiol (I) cleavage. It is remarkable that the bpa complex shows the highest yield of extradiol product and high product selectivity in aqueous SDS solution (E, 84.0%; E/I, 61.0 : 1) and in SDS : n-hexane reverse micellar medium (E, 93.7%) illustrating that a vacant or solvent coordinated site is essential for observing extradiol cleavage. Interestingly, the rates of dioxygenase reactions in aqueous and aqueous micellar solutions are significantly higher than those in non-aqueous solvents. Also, they diminish in the order, SDS > TX-100 > CTAB, illustrating the facile substitution of coordinated water molecule by molecular oxygen in [Fe(L)(DBC)(H(2)O)](+) bound to anionic SDS micelles.

Role of cis–cis muconic acid in the catalysis of Pseudomonas putida chlorocatechol 1,2-dioxygenase

Chlorocatechol 1,2-dioxygenase (1,2-CCD) is a non-heme iron protein involved in the intradiol cleavage of aromatic compounds that are recalcitrant to biodegradation. In particular, 1,2-CCD catalyzes the conversion of catechol and its halogenated derivatives to cis–cis muconic acid. In this study we describe a series of experiments concerning the interaction of chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp1,2-CCD) with cis–cis muconic acid. We used single-injection ITC to show that the reaction product inhibits enzyme kinetics. DSC and EPR measurements probed whether this was accomplished by a direct binding of the product to the enzyme active site. DSC shows that cis–cis muconic acid affects the thermal unfolding of the protein and allowed us to estimate a binding constant. Furthermore, EPR spectra of the Fe(III) center demonstrate that, upon product binding, a significant decrease in resonance intensity is observed, indicating that cis–cis muconic acid binds directly to the active site. Based on the increasing interest for understanding dioxygenases mechanism of action and, moreover, how to control such process, our data indicate that the product of the reaction does play a relevant role in the catalysis and should therefore be taken into account when one thinks about ways of regulating enzyme activity.