Reaction mechanism of the binuclear zinc enzyme glyoxalase II - A theoretical study

The glyoxalase system catalyzes the conversion of toxic methylglyoxal to nontoxic d-lactic acid using glutathione (GSH) as a coenzyme. Glyoxalase II (GlxII) is a binuclear Zn enzyme that catalyzes the second step of this conversion, namely the hydrolysis of S-d-lactoylglutathione, which is the product of the Glyoxalase I (GlxI) reaction. In this paper we use density functional theory method to investigate the reaction mechanism of GlxII. A model of the active site is constructed on the basis of the X-ray crystal structure of the native enzyme. Stationary points along the reaction pathway are optimized and the potential energy surface for the reaction is calculated. The calculations give strong support to the previously proposed mechanism. It is found that the bridging hydroxide is capable of performing nucleophilic attack at the substrate carbonyl to form a tetrahedral intermediate. This step is followed by a proton transfer from the bridging oxygen to Asp58 and finally C-S bond cleavage. The roles of the two zinc ions in the reaction mechanism are analyzed. Zn2 is found to stabilize the charge of tetrahedral intermediate thereby lowering the barrier for the nucleophilic attack, while Zn1 stabilizes the charge of the thiolate product, thereby facilitating the C-S bond cleavage. Finally, the energies involved in the product release and active-site regeneration are estimated and a new possible mechanism is suggested.     

Tungsten-dependent formaldehyde ferredoxin oxidoreductase: reaction mechanism from quantum chemical calculations

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a tungsten-dependent enzyme that catalyzes the oxidation of formaldehyde to formic acid. In the present study, quantum chemical calculations are used to elucidate the reaction mechanism of this enzyme. Several possible mechanistic scenarios are investigated with a large model of the active site designed on the basis of the X-ray crystal structure of the native enzyme. Based on the calculations, we propose a new mechanism in which the formaldehyde substrate binds directly to the tungsten ion. W^VI=O then performs a nucleophilic attack on the formaldehyde carbon to form a tetrahedral intermediate. In the second step, which is calculated to be rate limiting, a proton is transferred to the second-shell Glu308 residue, coupled with a two-electron reduction of the tungsten ion. The calculated barriers for the mechanism are energetically very feasible and in relatively good agreement with experimental rate constants. Three other second-shell mechanisms, including one previously proposed based on experimental findings, are considered but ruled out because of their high barriers.     

X-ray structure of isoaspartyl dipeptidase from E.coli: a dinuclear zinc peptidase evolved from amidohydrolases

L-aspartyl and L-asparaginyl residues in proteins spontaneously undergo intra-residue rearrangements forming isoaspartyl/beta-aspartyl residues linked through their side-chain beta-carboxyl group with the following amino acid. In order to avoid accumulation of isoaspartyl dipeptides left over from protein degradation, some bacteria have developed specialized isoaspartyl/beta-aspartyl zinc dipeptidases sequentially unrelated to other peptidases, which also poorly degrade alpha-aspartyl dipeptides. We have expressed and crystallized the 390 amino acid residue isoaspartyl dipeptidase (IadA) from E.coli, and have determined its crystal structure in the absence and presence of the phosphinic inhibitor Asp-Psi[PO(2)CH(2)]-LeuOH. This structure reveals an octameric particle of 422 symmetry, with each polypeptide chain organized in a (alphabeta)(8) TIM-like barrel catalytic domain attached to a U-shaped beta-sandwich domain. At the C termini of the beta-strands of the beta-barrel, the two catalytic zinc ions are surrounded by four His, a bridging carbamylated Lys and an Asp residue, which seems to act as a proton shuttle. A large beta-hairpin loop protruding from the (alphabeta)(8) barrel is disordered in the free peptidase, but forms a flap that stoppers the barrel entrance to the active center upon binding of the dipeptide mimic. This isoaspartyl dipeptidase shows strong topological homology with the alpha-subunit of the binickel-containing ureases, the dinuclear zinc dihydroorotases, hydantoinases and phosphotriesterases, and the mononuclear adenosine and cytosine deaminases, which all are catalyzing hydrolytic reactions at carbon or phosphorous centers. Thus, nature has adapted an existing fold with catalytic tools suitable for hydrolysis of amide bonds to the binding requirements of a peptidase.     

Mechanistic investigation of platinum-catalysed hydroformylation of propene: A density functional study

The mechanism of hydroformylation of propene in the presence of both cis-PtH(SnCl₃)(PH₃)₂ and trans-PtH(SnCl₃)(PH₃)₂ catalysts has been investigated. A density functional study has been carried out for all of the elementary steps of the catalytic cycle, i.e. for the alkene coordination, for its insertion into the Pt-H bond, carbon monoxide activation and its subsequent insertion into the Pt-alkyl bond. Finally, the product forming step, the dihydrogen activation and aldehyde elimination have been investigated.It has been found that the regioselectivity of hydroformylation is determined in the olefin insertion step. The computed ratio of the linear regioisomer, n-butanal is predicted to be 83% when solvation corrections were employed, being in very good agreement with the experimental result. Among the elementary steps the hydrogenolysis has been found to be the slowest followed by the migratory carbon monoxide insertion step. Due to the crucial role of the trichlorostannato ligand the electronic effects of SnCl₃ has been analysed employing the charge decomposition analysis (CDA), the natural bond orbital (NBO) and the atoms in molecules (AIM) methods.     

Quantum chemical modeling of enzymatic reactions: the case of 4-oxalocrotonate tautomerase

The reaction mechanism of 4-oxalocrotonate tautomerase (4-OT) is studied using the density functional theory method B3LYP. This enzyme catalyzes the isomerisation of unconjugated alpha-keto acids to their conjugated isomers. Two different quantum chemical models of the active site are devised and the potential energy curves for the reaction are computed. The calculations support the proposed reaction mechanism in which Pro-1 acts as a base to shuttle a proton from the C3 to the C5 position of the substrate. The first step (proton transfer from C3 to proline) is shown to be the rate-limiting step. The energy of the charge-separated intermediate (protonated proline-deprotonated substrate) is calculated to be quite low, in accordance with measured pKa values. The results of the two models are used to evaluate the methodology employed in modeling enzyme active sites using quantum chemical cluster models.     

DFT studies of the ionization of alpha and beta glycopyranosyl donors

Current attempts at mimicking the transition states (TSs) of glycosyl processing enzymes (GPEs) that proceed through TSs with a high degree of oxacarbenium ion formation suffer from a paucity of data about the conformations of such oxacarbenium ions. Because TSs are maxima, the current models based on minimized structures may need some refinement. As part of studies directed at optimizing chemical glycosylation the ionization of 3,4,6-tri-O-acetyl-alpha/beta-D-glucopyranosyl chlorides and triflates, 2,3,4,6-tetra-O-methyl-alpha/beta-D-glucopyranosyl fluorides, chlorides and triflates, 2,3,4,6-tetra-O-methyl-alpha/beta-D-mannopyranosyl fluorides, 2,3-di-O-methyl 4,6-O-benzylidene alpha/beta-D-mannopyranosyl triflates and 2,3-di-O-methyl 4,6-O-benzylidene alpha/beta-D-glucopyranosyl triflates was studied by a prototypic density functional theory (DFT) procedure. In all cases, the alpha-anomers ionized smoothly to 4H3 half chair conformations or adjacent envelopes. By contrast, all beta-anomers exhibited an abrupt conformational change before ionization was complete. The nature of the conformations sampled depends on both the leaving group and the protecting group. The methods presented can be readily adapted to the study of any GPE or chemical glycosylation and provide a method for initial evaluation of plausible TSs, which in turn can be used in mimetic design.     

DFT: a dynamic study of the interaction of ethanol and methanol with platinum

The interaction between alcohol molecules and platinum (Pt) was studied using molecular dynamics (MD; Born-Oppenheimer method). Alcohol molecules like ethanol and methanol present a similar molecular structure, with a methyl group (CH₃) at one end and a fragment of hydroxyl (OH) at the other. This fact generates two orientations that are considered in the interaction with Pt. The MD calculation results for these two orientations indicate a preferential orientation due to energy interactions. A plausible reaction mechanism that takes into account the interaction between Pt and alcohol is presented. The charge transference obtained from the Pt–alcohol interaction was also analyzed. The energy for the two orientations was calculated by indicating the preferential orientation. The methyl and hydroxyl groups are involved in heterolytic breakage of hydrogen bonds, joined to a carbon atom in the former and to an oxygen atom in the latter; however, the methyl group reaction seems to be the most important.     

The metal bonding domain of the antitumor drug Fe(II)-bleomycin: a DFT investigation

The geometric and electronic structure of ferrous complexes of bleomycin (Fe(II)BLM) has been investigated by means of density functional theory (DFT) calculations. The active site of this antitumor drug is a highly distorted octahedral complex, with the coordination sphere completed by the five known endogenous ligands, including pyrimidine, imidazole, deprotonated amide, and secondary and primary amines. We have addressed the controversial issue of the nature of the sixth axial ligand, which we have identified as the oxygen of the carbamoyl group. Our conclusions are further validated by a comparison with structural data derived from NMR experiments. Moreover, because of the high sensitivity of structural data on the pH of the environment, we have investigated the effect of a different protonation state of the histidine amide on the geometric structure of the Fe(II)BLM complex. The extensive model of the active site of bleomycin considered in this work allows us to check the limitations of previous investigations based on simplified models.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.An erratum to this article can be found at     

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of ¹⁵N-¹⁵N and ¹H-¹⁵N trans-hydrogen bond scalar couplings, ^2h J _NN and ^1h J _HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.     

Catecholase activity of dicopper(II)-bispidine complexes: stabilities and structures of intermediates,kinetics and reaction mechanism

A mechanism for the oxidation of 3,5-di-tert-butylcatechol (dtbc) with dioxygen to the corresponding quinone (dtbq), catalyzed by bispidine-dicopper complexes (bispidines are various mono- and dinucleating derivatives of 3,7-diazabicyclo[3.3.1]nonane with bis-tertiary-amine–bispyridyl or bis-tertiary-amine–trispyridyl donor sets), is proposed on the basis of (1) the stoichiometry of the reaction as well as the stabilities and structures [X-ray, density functional theory (B3LYP, TZV)] of the bispidine-dicopper(II)–3,4,5,6-tetrachlorcatechol intermediates, (2) formation kinetics and structures (molecular mechanics, MOMEC) of the end-on peroxo–dicopper(II) complexes and (3) kinetics of the stoichiometric (anaerobic) and catalytic (aerobic) copper-complex-assisted oxidation of dtbc. This involves (1) the oxidation of the dicopper(I) complexes with dioxygen to the corresponding end-on peroxo–dicopper(II) complexes, (2) coordination of dtbc as a bridging ligand upon liberation of H₂O₂ and (3) intramolecular electron transfer to produce dtbq, which is liberated, and the dicopper(I) catalyst. Although the bispidine complexes have reactivities comparable to those of recently published catalysts with macrocyclic ligands, which seem to reproduce the enzyme-catalyzed process in various reaction sequences, a strikingly different oxidation mechanism is derived from the bispidine–dicopper-catalyzed reaction. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Experimental determination and calculations of redox potential descriptors of compounds directed against retroviral zinc fingers: Implications for rational drug design.

A diverse set of electrophilic compounds that react with cysteine thiolates in retroviral nucleocapsid (NC) proteins and abolish virus infectivity has been identified. Although different in chemical composition, these compounds are all oxidizing agents that lead to the ejection of Zn(II) ions bound to conserved structural motifs (zinc fingers) present in retroviral NC proteins. The reactivity of a congeneric series of aromatic disulfides toward the NC protein of the human immunodeficiency virus type 1 (HIV-1), NCp7, has been characterized by HPLC separation of starting reagents from reaction products. We calculated the absolute redox potentials of these compounds in the gas phase and in aqueous solvent, using a density functional theory method and a continuum solvation model. Pulsed polarography experiments were performed and showed a direct correlation between calculated and experimentally determined redox propensities. A dependence between protein reactivity and redox potential for a specific compound was shown: Reaction with NCp7 did not take place below a threshold value of redox potential. This relationship permits the distinction between active and nonactive compounds targeted against NCp7, and provides a theoretical basis for a scale of reactivity with retroviral zinc fingers. Our results indicate that electrophilic agents with adequate thiophilicity to react with retroviral NC fingers can now be designed using known or calculated electrochemical properties. This may assist in the design of antiretroviral compounds with greater specificity for NC protein. Such electrophilic agents can be used in retrovirus inactivation with the intent of preparing a whole-killed virus vaccine formulation that exhibits unaffected surface antigenic properties.     

Noninnocent effect of axial ligand on the heme degradation process: a theoretical approach to hydrolysis pathway of verdoheme to biliverdin

Conversion of iron(II) verdoheme to iron(II) biliverdin in the presence of hydroxyl ion as a nucleophile and imidazole, pyridine, water, hydroxyl, cyanide, phenolate, chloride, thiolate and imidazolate as axial ligands was investigated using the B3LYP method and the 6-31G basis set. In the five-coordinated pathway the reactants and products are in the ground triplet state. In this path, hydroxyl ion directly attacks the macrocycle. The exothermic energy for addition of hydroxyl ion to iron(II) verdoheme with various ligands is 169.55, 166.34 and 164 kcal mol⁻¹ for water, pyridine and imidazole, energies which are around 30–60 kcal mol⁻¹ more exothermic than those for the other axial ligands used in this study. Therefore, imidazole, water and pyridine axial ligands can facilitate hydrolytic cleavage of iron(II) verdoheme to form open-chained helical iron(II) biliverdin complexes. The activation barrier for the conversion of iron(II) verdoheme hydroxyl species to the iron(II) biliverdin complex is estimated to be 5.2, 4.2, 4.35, 13.76 and 14.05 kcal mol⁻¹ for imidazole, water, cyanide, thiolate and imidazolate, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Investigation of intermolecular hydrogen bond interactions in crystalline l-Cysteine by DFT calculations of the oxygen-17, nitrogen-14, and hydrogen-2 EFG tensors and AIM analysis

A systematic computational study is carried out to investigate hydrogen bond (HB) interactions in the real crystalline structures of l-Cysteine at 30 and 298 K by density functional theory (DFT) calculations of electric field gradient (EFG) tensors at the sites of O-17, N-14, and H-2 nuclei. One-molecule (monomer) and nine-molecule (cluster) models of l-Cysteine are created by available crystal coordinates at both temperatures and the EFG tensors are calculated for both models to indicate the effect of HB interactions on the tensors. The calculated EFG tensors at the level of B3LYP and B3PW91 DFT methods and 6-311++G?? and cc-pVTZ basis sets are converted to those experimentally measurable nuclear quadrupole resonance (NQR) parameters i.e. quadrupole coupling constants (qcc) and asymmetry parameters (η_Q). The evaluated NQR parameters reveal that the EFG tensors of ¹⁷O, ¹⁴N, and ²H are influenced and show particular trends from monomer to the target molecule in the cluster due to the contribution of target molecule to classic N–H…O, and non-classic S–H…O and S–H…S types of HB interactions. On the other hand, atoms in molecules (AIM) analyses confirm the presence of HB interactions and rationalize the observed EFG trends. The results indicate different contribution of various nuclei to HB interactions in the cluster where O2 and N1 have major contributions. The EFG tensors as well as AIM analysis at the H6 site show that the N1-H6…O2 HB undergoes a significant change from 30 to 298 K where changes in other N–H…O interactions are almost negligible. There is a good agreement between the calculated ¹⁴N NQR parameters and reported experimental data.     

On the aggregation of bilirubinoids in solution as evidenced by VCD and ECD spectroscopy and DFT calculations

Vibrational and electronic circular dichroism (VCD and ECD) spectra of 3 optically active bilirubin analogs with propionic acid groups replaced by (1) 1‐(S)‐methylpropyl groups, (2) 3‐acetoxy‐1‐(S)‐methylpropyl groups, and (3) 1‐(S)‐2‐(R)‐dimethyl‐2‐(methoxycarbonyl)ethyl groups have been recorded at different concentrations in chloroform. The aliphatic chains attached to C‐8 and C‐12 of the 3 chosen mesobilirubins were modified so as to possess no OH group. The variation of the VCD spectra with concentration is consistent with the formation of dimers at high concentration. Density functional theory and time‐dependent density functional theory calculations on monomeric and dimeric forms support such a conclusion. Comparing with previous VCD (ECD) and IR (UV) studies of other mesobilirubin molecules, it is concluded that here, the key feature for aggregation is the missing OH groups on the propionic acid chains. The latter, in synergy with the polar groups of lactam moieties, appear to be involved in intramolecular phenomena and thus favor monomeric forms. Investigation of ECD and UV spectra of the same compounds in mixed DMSO/chloroform solutions provide further clues to the proposed picture.     

Theoretical study on electronic structures of FeOO, FeOOH, FeO(H2O), and FeO in hemes: as intermediate models of dioxygen reduction in cytochrome c oxidase

The electronic structures of heme-dioxygen complexes have been studied as intermediate models of dioxygen reduction mechanism catalyzed by the mixed valence (MV) and fully reduced (FR) cytochrome c oxidase (CcO). Dioxygen, protons and electrons were sequentially added to the heme along the proposed reaction path for the O(2) reduction mechanism. The electronic structures of [FeOO], [FeOO](-), [FeOOH](+), [FeOOH], [Fe=O, H(2)O](+), [Fe=O](+) and [Fe=O] were thoroughly investigated by using the unrestricted hybrid exchange-correlation functional B3LYP method. The additions of two protons and an electron to [FeOO] lead to the OO bond cleavage to produce a H(2)O molecule with the electron transfer from Fe(II) in heme to the OO moiety. It is shown that the intrinsic OO bond cleavage occurs by adding two protons and two electrons into the OO bond, indicating consistency with a H(2)O formation catalyzed by both MV and FR CcO. The study of the electronic structures of heme-dioxygen complexes gave the different proposals for the mechanisms of a H(2)O formation by both MV and FR CcO. For the mixed valence CcO, starting from the [FeOO] complex, the final products are single H(2)O molecule and compound I of the oxo heme. For the fully reduced CcO, the final products are single H(2)O molecule and compound II of the oxo heme which is a reduced state of the compound I.     

Structural model studies for the high-valent intermediate Q of methane monooxygenase from broken-symmetry density functional calculations

Mössbauer isomer shift parameters have been obtained for both density functional theory (DFT) OPBE and OLYP functionals by linear regressions between the measured isomer shifts and calculated electron densities at Fe nuclei for a number of Fe^2+,2.5+ and Fe^{2.5+,3+,3.5+,4+} complexes grouped separately. The calculated isomer shifts and quadrupole splittings on the sample Fe complexes from OPBE and OLYP functionals are similar to those of PW91 calculations [J. Comput. Chem. 27 (2006) 1292], however the fit parameters from the linear regressions differ between PW91 and OPBE, OLYP. Four models for the active site structure of intermediate Q of the hydroxylase component of soluble methane monooxygenase (MMOH) have been studied, using three DFT functionals OPBE, OLYP, and PW91, incorporated with broken-symmetry methodology and the conductor-like screening (COSMO) solvation model. The calculated properties, including optimized geometries, electronic energies, pK_a’s, Fe net spin populations, and Mössbauer isomer shifts and quadrupole splittings, have been reported and compared with available experimental values. The high-spin antiferromagnetically (AF) coupled Fe⁴⁺ sites are correctly predicted by OPBE and OLYP methods for all active site models. PW91 potential overestimates the Fe-ligand covalencies for some of the models because of spin crossover. Our calculations and data analysis support the structure (our current model II shown in Fig. 8) proposed by Friesner and Lippard’s group [J. Am. Chem. Soc. 123 (2001) 3836-3837], which contains an Fe⁴⁺(μ-O)₂Fe⁴⁺ center, one axial water which also H-bonds to both side chains of Glu243 and Glu114, and one bidentate carboxylate group from the side chain of Glu144, which is likely to represent the active site of MMOH-Q. A new model structure (model IV shown in Fig. 9), which has a terminal hydroxo and a protonated His147 which is dissociated from a nearby Fe, is more asymmetric in its Fe(μ-O)₂Fe diamond core, and is another very good candidate for intermediate Q.     

On the oxidation of alkyl and aryl sulfides by [(Me3TACN)MnO(OH)2]: A density functional study

Density functional theory suggests that the formal 2-electron oxidation of sulfides, RR′S, to sulfoxides, by the model Mn^VO catalyst, [(TACN)Mn^V O(OH)₂]⁺, proceeds in two quite distinct 1-electron steps. Transfer of the first electron is barrierless and generates a sulfur radical cation, antiferromagnetically coupled to a Mn^IV centre via a covalent μ-oxo bridge. The second electron-transfer step is accompanied by migration of the oxygen atom to the sulfur centre, and is rate-determining. The absence of a barrier in the first step, where a sulfur radical is formed, means that the presence of electron-donating or withdrawing substituents on the sulfide has only a minor impact on the rate of reaction.     

Lactating and nonlactating rats differ to renal toxicity induced by mercuric chloride: the preventive effect of zinc chloride

This study evaluated the effects of HgCl₂ on renal parameters in nonlactating and lactating rats and their pups, as well as the preventive role of ZnCl₂. Rats received 27 mg kg^?1 ZnCl₂ for five consecutive days and 5 mg kg^?1 HgCl₂ for five subsequent days (s.c.). A decrease in δ‐aminolevulinic acid dehydratase (δ‐ALA‐D) activity in the blood and an increase in urine protein content in renal weight as well as in blood and urine Hg levels were observed in lactating and nonlactating rats from Sal―Hg and Zn―Hg groups. ZnCl₂ prevented partially the δ‐ALA‐D inhibition and the proteinuria in nonlactating rats. Renal Hg levels were increased in all HgCl₂ groups, and the ZnCl₂ exposure potentiated this effect in lactating rats. Nonlactating rats exposed to HgCl₂ exhibited an increase in plasma urea and creatinine levels, δ‐ALA‐D activity inhibition and histopathological alterations (necrosis, atrophic tubules and collagen deposition) in the kidneys. ZnCl₂ exposure prevented the biochemical alterations. Hg‐exposed pups showed lower body and renal weight and an increase in the renal Hg levels. In conclusion, mercury‐induced nephrotoxicity differs considerably between lactating and nonlactating rats. Moreover, prior exposure with ZnCl₂ may provide protection to individuals who get exposed to mercury occupationally or accidentally. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of the absolute configurations of chiral organometallic complexes via density functional theory calculations of their vibrational circular dichroism spectra: The chiral chromium tricarbonyl complex of N-pivaloyl-tetrahydroquinoline

The racemate of the chiral tricarbonyl-η⁶-arene-chromium(0) complex, tricarbonyl-η⁶-N-pivaloyl-tetrahydroquinoline-chromium(0), 1, has been synthesized and resolved using chromatography on a (R,R)-Whelk-O1 column. The Absolute Configuration (AC) of 1 has been determined using vibrational circular dichroism (VCD) spectroscopy. The VCD spectrum of 1 has been predicted using the Stephens equation for vibrational rotational strengths, implemented using density functional theory (DFT) in the gaussian program. Using the B3PW91 functional and the 6-311++G (2d,2p) basis set, the predicted VCD spectrum of S-1 is in excellent agreement with the experimental VCD spectrum of (+)-1, leading unambiguously to the AC S-(+). It is concluded that VCD is a useful technique for determining the ACs of chiral organometallic complexes, given the use of optimum functionals and basis sets.