CD and MCD studies of the non-heme ferrous active site in (4-hydroxyphenyl)pyruvate dioxygenase: correlation between oxygen activation in the extradiol and alpha-KG-dependent dioxygenases

(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) is an unusual alpha-keto acid-dependent non-heme iron dioxygenase as it incorporates both atoms of dioxygen into a single substrate, paralleling the extradiol dioxygenases. CD/MCD studies of the catalytically active ferrous site and its interaction with substrate reveal a geometic and electronic structure and mechanistic approach to oxygen activation which bridges those of the alpha-KG-dependent and the extradiol dioxygenases.     

Spectroscopic studies of the anaerobic enzyme-substrate complex of catechol 1,2-dioxygenase

The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme/substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UV-vis) difference spectroscopic studies demonstrated that, in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that, in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm(-1) in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm(-1) in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. In illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution.     

Evidence from mechanistic probes for distinct hydroperoxide rearrangement mechanisms in the intradiol and extradiol catechol dioxygenases

Three mechanistic probes were used to investigate whether the Criegee rearrangement step of catechol 1,2-dioxygenase (CatA) from Acinetobacter sp. proceeds via a direct 1,2-acyl migration, via homolytic O-O cleavage, or via a benzene oxide-oxepin rearrangement. Incubation of CatA with 3-chloroperoxybenzoic acid led to the formation of a 9:1 mixture of 2-chlorophenol and 3-chlorophenol, via a mechanism involving O-O homolytic cleavage. Incubation of CatA with 2-hydroperoxy-2-methylcyclohexanone led to formation of 5,6-diketoheptan-1-ol, also consistent with an O-O homolytic cleavage mechanism, and not consistent with a direct 1,2-acyl migration. No reaction product was isolated from incubation of CatA with 6-hydroxymethyl-6-methylcyclohexa-2,4-dienone, an analogue that is able to undergo the benzene oxide-oxepin rearrangement, but not able to undergo O-O homolytic cleavage. In contrast, incubation of extradiol dioxygenase MhpB from Escherichia coli with 6-hydroxymethyl-6-methylcyclohexa-2,4-dienone led to the formation of a 2-tropolone ring expansion product, consistent with a direct 1,2-alkenyl migration for extradiol cleavage. Taken together, the results imply different mechanisms for the Criegee rearrangement steps of intradiol and extradiol catechol dioxygenases: a direct 1,2-alkenyl migration for extradiol cleavage and an O-O homolytic cleavage mechanism for intradiol cleavage.     

Novel iron(III) complexes of sterically hindered 4N ligands: regioselectivity in biomimetic extradiol cleavage of catechols

The iron(III) complexes of the 4N ligands 1,4-bis(2-pyridylmethyl)-1,4-diazepane (L1), 1,4-bis(6-methyl-2-pyridylmethyl)-1,4-diazepane (L2), and 1,4-bis(2-quinolylmethyl)-1,4-diazepane (L3) have been generated in situ in CH 3CN solution, characterized as [Fe(L1)Cl 2] (+) 1, [Fe(L2)Cl 2] (+) 2, and [Fe(L3)Cl 2] (+) 3 by using ESI-MS, absorption and EPR spectral and electrochemical methods and studied as functional models for the extradiol cleaving catechol dioxygenase enzymes. The tetrachlorocatecholate (TCC (2-)) adducts [Fe(L1)(TCC)](ClO 4) 1a, [Fe(L2)(TCC)](ClO 4) 2a, and [Fe(L3)(TCC)](ClO 4) 3a have been isolated and characterized by elemental analysis, absorption spectral and electrochemical methods. The molecular structure of [Fe(L1)(TCC)](ClO 4) 1a has been successfully determined by single crystal X-ray diffraction. The complex 1a possesses a distorted octahedral coordination geometry around iron(III). The two tertiary amine (Fe-N amine, 2.245, 2.145 A) and two pyridyl nitrogen (Fe-N py, 2.104, 2.249 A) atoms of the tetradentate 4N ligand are coordinated to iron(III) in a cis-beta configuration, and the two catecholate oxygen atoms of TCC (2-) occupy the remaining cis positions. The Fe-O cat bond lengths (1.940, 1.967 A) are slightly asymmetric and differ by 0.027 A only. On adding catecholate anion to all the [Fe(L)Cl 2] (+) complexes the linear tetradentate ligand rearranges itself to provide cis-coordination positions for bidentate coordination of the catechol. Upon adding 3,5-di- tert-butylcatechol (H 2DBC) pretreated with 1 equiv of Et 3N to 1- 3, only one catecholate-to-iron(III) LMCT band (648-800 nm) is observed revealing the formation of [Fe(L)(HDBC)] (2+) involving bidentate coordination of the monoanion HDBC (-). On the other hand, when H 2DBC pretreated with 2 equiv of Et 3N or 1 or 2 equiv of piperidine is added to 1- 3, two intense catecholate-to-iron(III) LMCT bands appear suggesting the formation of [Fe(L)(DBC)] (+) with bidentate coordination of DBC (2-). The appearance of the DBSQ/H 2DBC couple for [Fe(L)Cl 2] (+) at positive potentials (-0.079 to 0.165 V) upon treatment with DBC (2-) reveals that chelated DBC (2-) in the former is stabilized toward oxidation more than the uncoordinated H 2DBC. It is remarkable that the [Fe(L)(HDBC)] (2+) complexes elicit fast regioselective extradiol cleavage (34.6-85.5%) in the presence of O 2 unlike the iron(III) complexes of the analogous linear 4N ligands known so far to yield intradiol cleavage products exclusively. Also, the adduct [Fe(L2)(HDBC)] (2+) shows a higher extradiol to intradiol cleavage product selectivity ( E/ I, 181:1) than the other adducts [Fe(L3)(HDBC)] (2+) ( E/ I, 57:1) and [Fe(L1)(HDBC)] (2+) ( E/ I, 9:1). It is proposed that the coordinated pyridyl nitrogen abstracts the proton from chelated HDBC (-) in the substrate-bound complex and then gets displaced to facilitate O 2 attack on the iron(III) center to yield the extradiol cleavage product. In contrast, when the cleavage reaction is performed in the presence of a stronger base like piperidine or 2 equiv of Et 3N a faster intradiol cleavage is favored over extradiol cleavage suggesting the importance of bidentate coordination of DBC (2-) in facilitating intradiol cleavage.     

Modeling the 2-His-1-carboxylate facial triad: iron-catecholato complexes as structural and functional models of the extradiol cleaving dioxygenases

Mononuclear iron(II)- and iron(III)-catecholato complexes with three members of a new 3,3-bis(1-alkylimidazol-2-yl)propionate ligand family have been synthesized as models of the active sites of the extradiol cleaving catechol dioxygenases. These enzymes are part of the superfamily of dioxygen-activating mononuclear non-heme iron enzymes that feature the so-called 2-His-1-carboxylate facial triad. The tridentate, tripodal, and monoanionic ligands used in this study include the biologically relevant carboxylate and imidazole donor groups. The structure of the mononuclear iron(III)-tetrachlorocatecholato complex [Fe(L3)(tcc)(H2O)] was determined by single-crystal X-ray diffraction, which shows a facial N,N,O capping mode of the ligand. For the first time, a mononuclear iron complex has been synthesized, which is facially capped by a ligand offering a tridentate Nim,Nim,Ocarb donor set, identical to the endogenous ligands of the 2-His-1-carboxylate facial triad. The iron complexes are five-coordinate in noncoordinating media, and the vacant coordination site is accessible for Lewis bases, e.g., pyridine, or small molecules such as dioxygen. The iron(II)-catecholato complexes react with dioxygen in two steps. In the first reaction the iron(II)-catecholato complexes rapidly convert to the corresponding iron(III) complexes, which then, in a second slow reaction, exhibit both oxidative cleavage and auto-oxidation of the substrate. Extradiol and intradiol cleavage are observed in noncoordinating solvents. The addition of a proton donor results in an increase in extradiol cleavage. The complexes add a new example to the small group of synthetic iron complexes capable of eliciting extradiol-type cleavage and provide more insight into the factors determining the regioselectivity of the enzymes.     

A new tripodal iron(III) monophenolate complex: effects of ligand basicity, steric hindrance, and solvent on regioselective extradiol cleavage

The new iron(III) complex [Fe(L3)Cl(2)], where H(L3) is the tripodal monophenolate ligand N,N-dimethyl-N'-(pyrid-2-ylmethyl)-N'-(2-hydroxy-3,5-dimethylbenzyl)ethylenediamine, has been isolated and studied as a structural and functional model for catechol dioxygenase enzymes. The complex possesses a distorted octahedral iron(III) coordination geometry constituted by the phenolate oxygen, pyridine nitrogen and two amine nitrogens of the tetradentate ligand, and two cis-coordinated chloride ions. The Fe-O-C bond angle (134.0 degrees) and Fe-O bond length (1.889 Angstrom) are very close to those (Fe-O-C, 133 degrees and 148 degrees, Fe-O(tyrosinate), 1.81 and 1.91 Angstrom) of protocatechuate 3,4-dioxygenase enzymes. When the complex is treated with AgNO(3), the ligand-to-metal charge transfer (LMCT) band around 650 nm (epsilon, 2390 M(-1) cm(-1)) is red shifted to 665 nm with an increase in absorptivity (epsilon, 2630 M(-1) cm(-1)) and the Fe(III)/Fe(II) redox couple is shifted to a slightly more positive potential (-0.329 to -0.276 V), suggesting an increase in the Lewis acidity of the iron(III) center upon the removal of coordinated chloride ions. Furthermore, when 3,5-di-tert-butylcatechol (H(2)DBC) pretreated with 2 mol of Et(3)N is added to the complex [Fe(L3)Cl(2)] treated with 2 equiv of AgNO(3), two intense catecholate-to-iron(III) LMCT bands (719 nm, epsilon, 3150 M(-1) cm(-1); 494 nm, epsilon, 3510 M(-1) cm(-1)) are observed. Similar observations are made when H(2)DBC pretreated with 2 mol of piperidine is added to [Fe(L3)Cl(2)], suggesting the formation of [Fe(L3)(DBC)] with bidentate coordination of DBC(2-). On the other hand, when H(2)DBC pretreated with 2 mol of Et(3)N is added to [Fe(L3)Cl(2)], only one catecholate-to-iron(III) LMCT band (617 nm; epsilon, 4380 M(-1) cm(-1)) is observed, revealing the formation of [Fe(L3)(HDBC)(Cl)] involving monodentate coordination of the catecholate. The appearance of the DBSQ/H(2)DBC couple for [Fe(L3)(DBC)] at a potential (-0.083 V) more positive than that (-0.125 V) for [Fe(L3)(HDBC)(Cl)] reveals that chelated DBC(2-) in the former is stabilized toward oxidation more than the coordinated HDBC(-). It is remarkable that the complex [Fe(L3)(HDBC)(Cl)] undergoes slow selective extradiol cleavage (17.3%) of H(2)DBC in the presence of O(2), unlike the iron(III)-phenolate complexes known to yield only intradiol products. It is probable that the weakly coordinated (2.310 Angstrom) -NMe(2) group rather than chloride in the substrate-bound complex is displaced, facilitating O(2) attack on the iron(III) center and, hence, the extradiol cleavage. In contrast, when the cleavage reaction was performed in the presence of a stronger base-like piperidine before and after the removal of the coordinated chloride ions, a faster intradiol cleavage was favored over extradiol cleavage, suggesting the importance of the bidentate coordination of the catecholate substrate in facilitating intradiol cleavage. Also, intradiol cleavage is favored in dimethylformamide and acetonitrile solvents, with enhanced intradiol cleavage yields of 94 and 40%, respectively.     

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.     

Molecular Cloning and Characterization of a New Cold-active Extradiol Dioxygenase from a Metagenomic Library Derived from Polychlorinated Biphenyl-contaminated Soil

To find new extradiol dioxygenases(EDOs, EC 1.13.11.2), a metagenomics library was constructed from polychlorinated biphenyl-contaminated soil and was screened for some dioxygenase with aromatic ring cleavage activity. A novel EDO, designated as Bph­C_A, was identified and heterologously expressed in Escherichia coli. The deduced amino acid sequence of BphC­_A exhibited a homology of less than 60% with other known EDOs. Phylogenetic analysis of BphC­_A suggests that the protein is a novel member of the EDO family. The enzyme exhibits higher substrate affinity and catalytic efficiency toward 3-methylcatechol than toward 2,3-dihydroxybiphenyl or catechol, the preferred substrate of other known EDOs. The optimum activity of purified Bph­C_A occurred at pH=8.5 and 35℃, and Bph­C_A showed more than 40% of its initial activity at 5℃. The activity of purified Bph­C_A was significantly induced by Mn²⁺ and slightly reduced by Al³⁺, Cu²⁺ and Zn²⁺.     

Cysteine dioxygenase: structure and mechanism

Cysteine dioxygenase (CDO) catalyzes the oxidation of cysteine to cysteine sulfinic acid, which is the first major step in cysteine catabolism in mammalian tissues. Crystal structures of mouse, rat, human and bacterial CDO have recently become available and provide significant mechanistic insights. Unlike most non-heme Fe(II) dioxygenases, coordination of the Fe in CDO deviates from the 2-His-1-carboxylate facial triad archetype and instead adopts a His3 facial triad. This change is expected to have an influence on oxygen activation by the catalytic site. The structures also reveal the presence of a cysteinyltyrosine (Tyr157-Cys93) post-translational modification near the active site. Kinetic studies of mutant CDOs reveal that the cysteine residue is less critical than the tyrosine for enzyme activity. Inconsistencies about the details of the active site and the nature of substrate binding exist and are discussed. Herein we review the structural biology along with relevant kinetics studies that have been conducted on CDO for insights into the reaction mechanism of this novel non-heme iron dioxygenase.     

Quantum chemical studies of dioxygen activation by mononuclear non-heme iron enzymes with the 2-His-1-carboxylate facial triad

Density functional theory with the B3LYP hybrid functional has been used to study the mechanisms for dioxygen activation by four families of mononuclear non-heme iron enzymes: alpha-ketoacid-dependent dioxygenases, tetrahydrobiopterin-dependent hydroxylases, extradiol dioxygenases, and Rieske dioxygenases. These enzymes have a common active site with a ferrous ion coordinated to two histidines and one carboxylate group (aspartate or glutamate). In contrast to the heme case, this type of weak field environment always leads to a high-spin ground state. With the exception of the Rieske dioxygenases, which have an electron source outside the active site, the dioxygen activation process passes through the formation of a bridging-peroxide species, which then undergoes O-O bond cleavage finally leading to the four electron reduction of O(2). In the case of tetrahydrobiopterin- and alpha-ketoacid-dependent enzymes, the O-O heterolysis yields a high-valent iron-oxo species, which is capable of performing a two-electron oxidation chemistry on various organic substrates. For the other two families of enzymes (extradiol dioxygenases and Rieske dioxygenases) the substrate oxidation and the O-O bond cleavage are found to be coupled. In the extradiol dioxygenases the product of the O-O bond cleavage is a ferric iron with an oxy-substrate with a mixture of radical and anionic character, which is essential for the selectivity of the catechol cleavage.     

Models for extradiol cleaving catechol dioxygenases: syntheses, structures, and reactivities of iron(II)-monoanionic catecholate complexes

Crystallographic and spectroscopic studies of extradiol cleaving catechol dioxygenases indicate that the enzyme-substrate complexes have both an iron(II) center and a monoanionic catecholate. Herein we report a series of iron(II)-monoanionic catecholate complexes, [(L)Fe(II)(catH)](X) (1a, L = 6-Me(3)-TPA (tris(6-methyl-2-pyridylmethyl)amine), catH = CatH (1,2-catecholate monoanion); 1b, L = 6-Me(3)-TPA, catH = DBCH (3,5-di-tert-butyl-1,2-catecholate monoanion); 1c, L = 6-Me(2)-bpmcn (N,N'-dimethyl-N,N'-bis(6-methyl-2-pyridylmethyl)-trans-1,2-diaminocyclohexane), catH = CatH; 1d, L = 6-Me(2)-bpmcn, catH = DBCH), that model such enzyme complexes. The crystal structure of [(6-Me(2)-bpmcn)Fe(II)(DBCH)](+) (1d) shows that the DBCH ligand binds to the iron asymmetrically as previously reported for 1b, with two distinct Fe-O bonds of 1.943(1) and 2.344(1) A. Complexes 1 react with O(2) or NO to afford blue-purple iron(III)-catecholate dianion complexes, [(L)Fe(III)(cat)](+) (2). Interestingly, crystallographically characterized 2d, isolated from either reaction, has the N-methyl groups in a syn configuration, in contrast to the anti configuration of the precursor complex, so epimerization of the bound ligand must occur in the course of isolating 2d. This notion is supported by the fact that the UV-vis and EPR properties of in situ generated 2d(anti) differ from those of isolated 2d(syn). While the conversion of 1 to 2 in the presence of O(2) occurs without an obvious intermediate, that in the presence of NO proceeds via a metastable S = (3)/(2) [(L)Fe(catH)(NO)](+) adduct 3, which can only be observed spectroscopically but not isolated. Intermediates 3a and 3b subsequently disproportionate to afford two distinct complexes, [(6-Me(3)-TPA)Fe(III)(cat)](+) (2a and 2b) and [(6-Me(3)-TPA)Fe(NO)(2)](+) (4) in comparable yield, while 3d converts to 2d in 90% yield. Complexes 2b and anti-2d react further with O(2) over a 24 h period and afford a high yield of cleavage products. Product analysis shows that the products mainly derive from intradiol cleavage but with a small extent of extradiol cleavage (89:3% for 2b and 78:12% for anti-2d). The small amounts of the extradiol cleavage products observed may be due to the dissociation of an alpha-methyl substituted pyridyl arm, generating a complex with a tridentate ligand. Surprisingly, syn-2d does not react with O(2) over the course of 4 days. These results suggest that there are a number of factors that influence the mode and rate of cleavage of catechols coordinated to iron centers.     

Mechanism for catechol ring cleavage by non-heme iron intradiol dioxygenases: a hybrid DFT study

The mechanism of the catalytic reaction of protocatechuate 3,4-dioxygenase (3,4-PCD), a representative intradiol dioxygenase, was studied with the hybrid density functional method B3LYP. First, a smaller model involving only the iron first-shell ligands (His460, His462, and Tyr408) and the substrates (catechol and dioxygen) was used to probe various a priori plausible reaction mechanisms. Then, an extended model involving also the most important second-shell groups (Arg457, Gln477, and Tyr479) was used for the refinement of the preselected mechanisms. The computational results suggest that the chemical reactions constituting the catalytic cycle of intradiol dioxygenases involve: (1) binding of the substrate as a dianion, in agreement with experimental suggestions, (2) binding of dioxygen to the metal aided by an electron transfer from the substrate to O(2), (3) formation of a bridging peroxo intermediate and its conformational change, which opens the coordination site trans to His462, (4) binding of a neutral XOH ligand (H(2)O or Tyr447) at the open site, (5) proton transfer from XOH to the neighboring peroxo ligand yielding the hydroperoxo intermediate, (6) a Criegee rearrangement leading to the anhydride intermediate, and (7) hydrolysis of the anhydride to the final acyclic product. One of the most important results obtained is that the Criegee mechanism requires an in-plane orientation of the four atoms (two oxygen and two carbon atoms) mainly involved in the reaction. This orientation yields a good overlap between the two sigma orbitals involved, C-C sigma and O-O sigma, allowing an efficient electron flow between them. Another interesting result is that under some conditions, a homolytic O-O bond cleavage might compete with the Criegee rearrangement. The role of the second-shell residues and the substituent effects are also discussed.     

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 (k _cat =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 (k _obs =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.     

Catalytic reaction mechanism of homogentisate dioxygenase: a hybrid DFT study

Human homogentisate dioxygenase is an Fe(II)-dependent enzyme responsible for aromatic ring cleavage. The mechanism of its catalytic reaction has been investigated with the hybrid density functional method B3LYP. A relatively big model of the active site was first used to determine the substrate binding mode. It was found that binding of the substrate dianion with a vacant position trans to Glu341 is most favorable. The model was then truncated to include only the most relevant parts of the active-site residues involved in iron coordination and substrate binding. Thus, methylimidazole was used to model His292, His335, His365, and His371, while propionate modeled Glu341. The computational results suggest that the catalytic reaction of homogentisate dioxygenases involves three major chemical steps: formation of the peroxo intermediate, homolytic cleavage of the O-O bond leading to an arene oxide radical, and finally, cleavage of the six-membered ring. Calculated barriers for alternative reaction paths are markedly higher than for the proposed mechanism, and thus the computational results successfully explain the product specificity of the enzyme. Interestingly, the results indicate that the type of ring scission, intra or extra with respect to the substituents coordinating to iron, is controlled by the barrier heights for the decay of the arene oxide radical intermediate.     

Extradiol oxidative cleavage of catechols by ferrous and ferric complexes of 1,4,7-triazacyclononane: insight into the mechanism of the extradiol catechol dioxygenases.

The major oxygenation product of catechol by dioxygen in the presence of FeCl(2) or FeCl(3), 1,4,7-triazacyclononane (TACN), and pyridine in methanol is the extradiol cleavage product 2-hydroxymuconic semi-aldehyde methyl ester (Lin, G.; Reid, G.; Bugg, T. D. H. J. Chem. Soc. Chem. Commun. 2000, 1119--1120). Under these conditions, extradiol cleavage of a range of 3- and 4-substituted catechols with electron-donating substituents is observed. The reaction shows a preference in selectivity and rate for iron(II) rather than iron(III) for the extradiol cleavage, which parallels the selectivity of the extradiol dioxygenase family. The reaction also shows a high selectivity for the macrocyclic ligand, TACN, over a range of other nitrogen- and oxygen-containing macrocycles. Reaction of anaerobically prepared iron-TACN complexes with dioxygen gave the same product as monitored by UV/vis spectroscopy. KO(2) is able to oxidize catechols with both electron-donating and electron-withdrawing substituents, implying a different mechanism for extradiol cleavage. Saturation kinetics were observed for catechols, which fit the Michaelis--Menten equation to give k(cat)(app) = 4.8 x 10(-3) s(-1) for 3-(2',3'-dihydroxyphenyl)propionic acid. The reaction was also found to proceed using monosodium catecholate in the absence of pyridine, but with different product ratios, giving insight into the acid/base chemistry of extradiol cleavage. In particular, extradiol cleavage in the presence of iron(II) shows a requirement for a proton donor, implying a role for an acidic group in the extradiol dioxygenase active site.     

Characterization of an O2 adduct of an active cobalt-substituted extradiol-cleaving catechol dioxygenase

The first example of an O(2) adduct of an active Co-substituted oxygenase has been observed in the extradiol ring cleavage of the electron-poor substrate 4-nitrocatechol (4NC) by Co(II)-homoprotocatechuate 2,3-dioxygenase (Co-HPCD). Upon O(2) binding to the high-spin Co(II) (S = (3)/(2)) enzyme-substrate complex, an S = (1)/(2) EPR signal exhibiting (59)Co hyperfine splitting (A = 24 G) typical of a low-spin Co(III)-superoxide complex was observed. Both the formation and decay of the new intermediate are very slow in comparison to the analogous steps for turnover of 4NC by native high-spin Fe(II)-HPCD, which is likely to remain high-spin upon O(2) binding. A similar but effectively stable S = (1)/(2) intermediate was formed by the inactive [H200N-Co-HPCD(4NC)] variant. The observations presented shed light on the key roles played by the substrate, the second-sphere His200 residue, and the spin state of the metal center in facilitating O(2) binding and activation.     

Iron(III) complexes of tridentate 3N ligands as functional models for catechol dioxygenases: the role of ligand N-alkyl substitution and solvent on reaction rate and product selectivity

A series of iron(III) complexes of the type [Fe(L)Cl3], where L is the variously N-alkyl-substituted bis(pyrid-2-ylmethyl)amine ligand such as bis(pyrid-2-ylmethyl)amine (L1), N,N-bis(pyrid-2-ylmethyl)methylamine (L2), N,N-bis(pyrid-2-ylmethyl)-n-propylamine (L3), N,N-bis(pyrid-2-ylmethyl)-iso-butylamine (L4), N,N-bis(pyrid-2-ylmethyl)-iso-propylamine (L5), N,N-bis(pyrid-2-ylmethyl)cyclohexylamine (L6), and N,N-bis(pyrid-2-ylmethyl)-tert-butylamine (L7), have been isolated and characterized by elemental analysis and spectral and electrochemical methods. The crystal structures of the complexes [Fe(L2)Cl3] 2, [Fe(L3)Cl3] 3, and the complex-substrate adduct [Fe(L5)(TCC)(NO3)] 5a, where TCC2- is the tetrachlorocatecholate dianion, have been determined by single-crystal X-ray crystallography. The complexes [Fe(L2)Cl3] 2 and [Fe(L3)Cl3] 3 possess a distorted octahedral geometry, in which the linear tridentate 3N ligands are cis-facially coordinated to the iron(III) center, and three chloride ions occupy the remaining coordination sites. The replacement of the N-methyl group in 2 by N-n-propyl group as in 3 leads to the formation of the Fe-Npy bonds and also the Fe-Cl bonds located trans to them of different lengths. The catecholate adduct 5a also possesses a distorted octahedral geometry, in which the ligand is cis-facially coordinated to iron(III) center, TCC2- is asymmetrically chelated trans to the two pyridyl moieties of the ligand, and one of the oxygen atoms of the nitrate ion occupies the sixth coordination site. All of the present complexes have been interacted with simple and substituted catechols. The catecholate adducts [Fe(L)(DBC)Cl] and [Fe(L)(DBC)(Sol)]+, where H2DBC is 3,5-di-tert-butylcatechol and Sol=H2O/CH3CN, have been generated in situ, and their spectral and redox properties and dioxygenase activities have been studied in dimethylformamide and dichloromethane solutions. All of the complexes catalyze the cleavage of H2DBC using molecular oxygen to afford both intra- and extradiol cleavage products. The formation of extradiol cleavage products is facilitated by cis-facial coordination of the 3N ligands and availability of vacant coordination site on iron(III) center for dioxygen binding. It is remarkable that the nature of the N-alkyl substituent in 3N ligands controls the regioselectivity of cleavage, with the n-propyl, iso-butyl, iso-propyl, and cyclohexyl groups enhancing the yield of extradiol products (46-68%) in dichloromethane. The rate of oxygenation depends upon the solvent and the Lewis acidity of iron(III) center as modified by the sterically demanding N-alkyl groups-length and degree of substitution. The plot of log (kO2) versus energy of the low-energy DBC2--to-iron(III) LMCT band is linear, demonstrating the importance of the Lewis acidity of the iron(III) center in dictating the rate of the dioxygenase reaction.     

Mimicking the Aromatic‐Ring‐Cleavage Activity of Gentisate‐1,2‐Dioxygenase by a Nonheme Iron Complex

Gentisate‐1,2‐dioxygenase (GDO), a nonheme iron enzyme in the cupin superfamily, catalyzes the cleavage of the aromatic‐ring of 2,5‐dihydroxybenzoic acid (gentisic acid) to form maleylpyruvic acid in the microbial aerobic degradation of aromatic compounds. To develop a functional model of GDO, we have isolated a nonheme iron(II) complex, [(Tp^Ph2)Fe^II(DHN‐H)] (Tp^Ph2=hydrotris(3,5‐diphenylpyrazole‐1‐yl)borate, DHN‐H=1,4‐dihydroxy‐2‐naphthoate). In the reaction with O₂, the biomimetic complex oxidatively cleaves the aromatic ring of the coordinated substrate with the incorporation of both the oxygen atoms from molecular oxygen into the cleavage product. The presence of para‐hydroxy group on the substrate plays a crucial role in directing the aromatic‐ring cleaving reaction.     

Electrostatic Perturbations from the Protein Affect C−H Bond Strengths of the Substrate and Enable Negative Catalysis in the TmpA Biosynthesis Enzyme

The nonheme iron dioxygenase 2-(trimethylammonio)-ethylphosphonate dioxygenase (TmpA) is an enzyme involved in the regio- and chemoselective hydroxylation at the C¹-position of the substrate as part of the biosynthesis of glycine betaine in bacteria and carnitine in humans. To understand how the enzyme avoids breaking the weak C²−H bond in favor of C¹-hydroxylation, we set up a cluster model of 242 atoms representing the first and second coordination sphere of the metal center and substrate binding pocket, and investigated possible reaction mechanisms of substrate activation by an iron(IV)-oxo species by density functional theory methods. In agreement with experimental product distributions, the calculations predict a favorable C¹-hydroxylation pathway. The calculations show that the selectivity is guided through electrostatic perturbations inside the protein from charged residues, external electric fields and electric dipole moments. In particular, charged residues influence and perturb the homolytic bond strength of the C¹−H and C²−H bonds of the substrate, and strongly strengthens the C²−H bond in the substrate-bound orientation.     

Iron(III) complexes of sterically hindered tetradentate monophenolate ligands as functional models for catechol 1,2-dioxygenases: the role of ligand stereoelectronic properties

The iron(III) complexes of the monophenolate ligands 2-(bis(pyrid-2-ylmethyl)aminomethyl)-4-nitrophenol [H(L1)], N,N-dimethyl-N'-(pyrid-2-ylmethyl)-N'-(2-hydroxy-4-nitrobenzyl)ethylenediamine [H(L2)], N,N-dimethyl-N'-(6-methyl-pyrid-2-ylmethyl)-N'-(2-hydroxy-4-nitrobenzyl)ethylenediamine [H(L3)], and N,N-dimethyl-N'-(1-methylimidazole-2-ylmethyl)-N'-(2-hydroxy-4-nitrobenzyl)ethylenediamine [H(L4)] have been obtained and studied as structural and functional models for the intradiol-cleaving catechol dioxygenase enzymes. The complexes [Fe(L1)Cl(2)].CH(3)CN (1), [Fe(L2)Cl(2)] (2), [Fe(L3)Cl(2)] (3), and [Fe(L4)Cl(2)] (4) have been characterized using absorption spectral and electrochemical methods. The single crystal X-ray crystal structures of 1 and 2 have been successfully determined. Both the complexes possess a rhombically distorted octahedral coordination geometry for the FeN(3)OCl(2) chromophore. In 2, the phenolate oxygen, the pyridine nitrogen, an amine nitrogen, and a chloride ion are located on the corners of a square plane with the nitrogen atom of a -NMe(2) group and the other chloride ion occupying the axial positions. In 1, also the equatorial plane is constituted by the phenolate oxygen, the pyridine nitrogen, an amine nitrogen atom, and a chloride ion; however, the axial positions are occupied by the second pyridine nitrogen and the second chloride ion. Interestingly, the Fe-O-C angle of 136.1 degrees observed for 2 is higher than that (128.5 degrees ) in 1; however, the Fe-O(phenolate) bond distances in both the complexes are the same (1.929 A). This illustrates the importance of the nearby sterically demanding coordinated -NMe(2) group and implies similar stereochemical constraints from the other ligated amino acid moieties in the 3,4-PCD enzymes, the enzyme activity of which is traced to the difference in the equatorial and axial Fe-O(tyrosinate) bonds (Fe-O-C, 133 degrees, 148 degrees ). The nature of heterocyclic rings of the ligands and the methyl substituents on them regulates the electronic spectral features, Fe(III)/Fe(II) redox potentials, and catechol cleavage activity of the complexes. Upon interacting the complexes with catecholate anions, two catecholate to iron(III) charge transfer bands appear, and the low energy band is similar to that of catechol dioxygenase-substrate complex. Complexes 1 and 3 fail to catalyze the oxidative intradiol cleavage of 3,5-di-tert-butylcatechol (H(2)DBC). However, interestingly, the replacement of pyridine pendant in 1 by the -NMe(2) group to obtain 2 restores the dioxygenase activity, which is consistent with its higher Fe-O-C bond angle. Remarkably, the more basic N-methylimidazole ring in 4 facilitates the rate-determining product releasing phase of the catalytic reaction, leading to enhancement in reaction rate and efficient conversion (77.1%) of the substrate to intradiol cleavage products as well. All these observations provide support to the novel substrate activation mechanism proposed for the intradiol-cleavage pathway.