Direct detection of oxygen intermediates in the non-heme Fe enzyme taurine/alpha-ketoglutarate dioxygenase

The reaction of substrate-bound taurine/alpha-ketoglutarate dioxygenase with O2 has been studied using cryogenic continuous-flow spectroscopy. Transient absorption spectra acquired at -38 degrees C show an exponential decay of a 318-nm chromophore with an apparent rate of 1.3 s-1. The observed optical changes and their kinetics are consistent with the profile of an Fe(IV) species detected recently by M?ssbauer spectroscopy (Price et al., Biochemistry 2003, 42, 7497-7508). Resonance Raman measurement upon excitation at 363.7 nm reveal at least two oxygen isotope-sensitive vibrations at 821/787 cm-1 and 583/555 cm-1 for 16O and 18O derivatives, respectively. An additional mode is likely to be obscured by an ethylene glycol vibration at 865 cm-1 and/or 1089 cm-1. The 821 cm-1 vibration is assigned to the stretching mode of Fe(IV)=O species on the basis of its frequency and isotopic shift amplitude. The 583 cm-1 band is likely to originate from an Fe-O2 precursor of the Fe(IV)=O species, although its structural details are unclear at present.     

Comparative quantum mechanics/molecular mechanics (QM/MM) and density functional theory calculations on the oxo-iron species of taurine/alpha-ketoglutarate dioxygenase

We present here the first quantum mechanical/molecular mechanics (QM/MM) studies of taurine/alpha-ketoglutarate dioxygenase (TauD) enzymes. Our studies are focused on the chemical properties of the oxo-iron species and the effect of the protein environment on its structural and electronic behavior. Although the active site region of TauD is very polar with many key hydrogen bonding interactions and salt bridges, the actual effect of the protein environment on the ordering and relative energies of the possible spin state structures is found to be quite small. Optimized geometries are very close to ones observed with density functional theory models that did not take the protein environment into consideration. The calculations show that protonation of the histidine ligands of iron is essential to reproduce the correct electronic representations of the enzyme. Hydroxylation studies of taurine by the oxo-iron active species predict that it is a very efficient catalyst that reacts with substrates via low reaction barriers.     

Evidence for hydrogen abstraction from C1 of taurine by the high-spin Fe(IV) intermediate detected during oxygen activation by taurine:alpha-ketoglutarate dioxygenase (TauD)

The Fe(II)- and alpha-ketoglutarate-dependent dioxygenases catalyze hydroxylation reactions of considerable biomedical and environmental significance. Recently, the first oxidized iron intermediate in the reaction of a member of this family, taurine:alpha-ketoglutarate dioxygenase (TauD), was detected and shown to be a high-spin, formally Fe(IV) complex. The demonstration in this study that decay of the Fe(IV) complex is approximately 30-fold slower when it is formed in the presence of 1-[2H]2-taurine provides evidence that the intermediate abstracts hydrogen from C1, the site of hydroxylation, and suggests that quantum-mechanical tunneling may contribute to C1-H cleavage.     

EXAFS spectroscopic evidence for an Fe=O unit in the Fe(IV) intermediate observed during oxygen activation by taurine:alpha-ketoglutarate dioxygenase

The Fe(II)- and alpha-ketoglutarate-dependent dioxygenases catalyze hydroxylation reactions of considerable biomedical and environmental significance. Recently, the first oxidized iron intermediate in the reaction of a member of this family, taurine:alpha-ketoglutarate dioxygenase (TauD), was detected and shown to be a high-spin Fe(IV) complex. In this study we have used X-ray absorption spectroscopy to demonstrate the presence of a short (1.62 A) interaction between the iron and one of its ligands in the Fe(IV) intermediate but not in the Fe(II) starting complex. The detection of this interaction strongly corroborates the hypothesis that the intermediate contains an Fe=O structural motif.     

Theoretical investigation on transformation of Cr(II) to Cr(V) complexes bearing tetra-N-heterocyclic carbene and group transfer reactivity

The mechanism for the transformation among a series of Cr(II) to Cr(V) complexes bearing tetra-N-heterocyclic carbene macrocycle is investigated. The oxidation and aziridination of Cr(II) monomer are studied by local density functional M06L. The former generates Cr(IV) oxo and further oxidizes to cationic Cr(V) oxo. The latter proceeds via two paths with different multiplicity, forming Cr(IV) imide. The Cr(IV) oxo cannot transfer its oxygen atom neither to phosphine nor to alkene because of the high energy barrier and endothermic process. The group transfer reactions are explored for Cr(V) oxo and Cr(IV) imide. The doublet Cr(V) oxo can also exist as quartet Cr(IV)-oxyl radical and promote oxygen transfer to phosphine, resulting in phosphine oxide in one exergonic step. The macrocyclic ligand effect of imido group transfer from Cr(IV) imide is verified by Multiwfn analysis. For 18-ringed imide, the matched orbital type and same-phase overlap reduce the barrier of its 16-ringed analog and facilitate the formation of phosphorus imine.     

Spectroscopic and computational evaluation of the structure of the high-spin Fe(IV)-oxo intermediates in taurine: alpha-ketoglutarate dioxygenase from Escherichia coli and its His99Ala ligand variant

The Fe(II)- and alpha-ketoglutarate (alphaKG)-dependent dioxygenases activate O2 for cleavage of unactivated C-H bonds in their substrates. The key intermediate that abstracts hydrogen in the reaction of taurine:alphaKG dioxygenase (TauD), a member of this enzyme family, was recently characterized. The intermediate, denoted J, was shown to contain an iron(IV)-oxo unit. Other important structural features of J, such as the number, identity, and disposition of ligands in the Fe(IV) coordination sphere, are not yet understood. To probe these important structural features, a series of models for J with the Fe(IV) ion coordinated by the expected two imidazole (from His99 and His255), two carboxylate (succinate and Asp101), and oxo ligands have been generated by density functional theory (DFT) calculations, and spectroscopic parameters (M?ssbauer isomer shift, quadrupole splitting, and asymmetry parameter, 57Fe hyperfine coupling tensor, and zero field splitting parameters, D and E/D) have been calculated for each model. The calculated parameters of distorted octahedral models for J, in which one of the carboxylates serves as a monodentate ligand and the other as a bidentate ligand, and a trigonal bipyramidal model, in which both carboxylates serve as monodentate ligands, agree well with the experimental parameters, whereas the calculated parameters of a square pyramidal model, in which the oxo ligand is in the equatorial plane, are inconsistent with the data. Similar analysis of the Fe(IV) complex generated in the variant protein with His99, the residue that contributes the imidazole ligand cis to the oxo group, replaced by alanine suggests that the deleted imidazole is replaced by a water ligand. This work lends credence to the idea that the combination of M?ssbauer spectroscopy and DFT calculations can provide detailed structural information for reactive intermediates in the catalytic cycles of iron enzymes.     

Scrutinizing low-spin Cr(II) complexes

The oxidation state of the chromium center in the following compounds has been probed using a combination of chromium K-edge X-ray absorption spectroscopy and density functional theory: [Cr(phen)(3)][PF(6)](2) (1), [Cr(phen)(3)][PF(6)](3) (2), [CrCl(2)((t)bpy)(2)] (3), [CrCl(2)(bpy)(2)]Cl(0.38)[PF(6)](0.62) (4), [Cr(TPP)(py)(2)] (5), [Cr((t)BuNC)(6)][PF(6)](2) (6), [CrCl(2)(dmpe)(2)] (7), and [Cr(Cp)(2)] (8), where phen is 1,10-phenanthroline, (t)bpy is 4,4'-di-tert-butyl-2,2'-bipyridine, and TPP(2-) is doubly deprotonated 5,10,15,20-tetraphenylporphyrin. The X-ray crystal structures of complexes 1, [Cr(phen)(3)][OTf](2) (1'), and 3 are reported. The X-ray absorption and computational data reveal that complexes 1-5 all contain a central Cr(III) ion (S(Cr) = (3)/(2)), whereas complexes 6-8 contain a central low-spin (S = 1) Cr(II) ion. Therefore, the electronic structures of 1-8 are best described as [Cr(III)(phen(?))(phen(0))(2)][PF(6)](2), [Cr(III)(phen(0))(3)][PF(6)](3), [Cr(III)Cl(2)((t)bpy(?))((t)bpy(0))], [Cr(III)Cl(2)(bpy(0))(2)]Cl(0.38)[PF(6)](0.62), [Cr(III)(TPP(3?-))(py)(2)], [Cr(II)((t)BuNC)(6)][PF(6)](2), [Cr(II)Cl(2)(dmpe)(2)], and [Cr(II)(Cp)(2)], respectively, where (L(0)) and (L(?))(-) (L = phen, (t)bpy, or bpy) are the diamagnetic neutral and one-electron-reduced radical monoanionic forms of L, and TPP(3?-) is the one-electron-reduced doublet form of diamagnetic TPP(2-). Following our previous results that have shown [Cr((t)bpy)(3)](2+) and [Cr(tpy)(2)](2+) (tpy = 2,2':6',2"-terpyridine) to contain a central Cr(III) ion, the current results further refine the scope of compounds that may be described as low-spin Cr(II) and reveal that this is a very rare oxidation state accessible only with ligands in the strong-field extreme of the spectrochemical series.     

Prediction of Stability Constants for Cr(III) and Cr(II) Complexes

Regression analysis was used to derive equations for estimaing thermodynamic stability constants for complexes of Cr²⁺ (log^° ₁[Cr²⁺L] = 0.53log^° _n [H _n L]) and Cr³⁺ (log^° ₁[Cr³⁺L] = 0.88log^° _n [H _n L]) from the known protonation constants of H _n L ligands and for determining stability constants of Cr²⁺ and Cr³⁺ complexes from the available stability constants of Cu²⁺ complexes (log^° ₁[Cr²⁺L] = 0.76log^° ₁[Cu²⁺L] and log^° ₁[Cr²⁺L] = 0.60log^° ₁[Cr³⁺L], respectively). Parameters of the Panteleon–Ecka equation for calculating stability constants of Cr²⁺ complexes ( = 0.57) and Cr³⁺ complexes ( = 0.69) with two and three bidentate ligands were also determined. The ratio of logarithmic stability constants for complexes with the same metals but with different metal ionic charges was found to be approximately equal to the ratio of charges on the central ions. The stability constant of Cr(II) sulfate complex was calculated.     

Electrode processes in the course of Cr(III) and Cr(II) reduction/oxidation in KCl melt

A hypothesis about formation of a polynuclear chromium complex is made on the basis of a voltammetric study of the processes of trivalent and divalent chromium reduction within complex chloride ions in a potassium chloride melt. The effect of the reaction of Cr₂Cl ₇ ^3? formation on deviation of theoretical dependences from the experimentally obtained dependences under the terms of linear voltammetry is shown. Models are suggested for the complex chromium ions, quantum-chemical calculations of the bond lengths in them are performed and steric energies of ions are determined. Diffusion coefficients of Cr(III) and Cr(II) are calculated, diffusion activation energies are determined.     

Insights on Rh(II) carbenoid reactivity

A computational study on a range of Rh(II) carbenoids shows how carbenoid stability and cyclopropanation diastereoselectivity can be affected by certain properties of the carbenoid substituents. The results of the study imply that substituents capable of π-interactions are stabilising and cis-directing, and that the trans-directing abilities are affected by steric effects as well as the polarity of carbonyl groups.     

Comparative sequence analysis and mutagenesis of Ethylene Forming Enzyme (EFE) 2-oxoglutarate/Fe(II)-dependent dioxygenase homologs

Background

Ethylene is one of the most used chemical monomers derived from non-renewable sources and we are investigating the possibility of producing it in yeast via the ethylene forming enzyme (EFE) from Pseudomonas syringae. To enable engineering strategies to improve the enzyme, it is necessary to identify the regions and amino acid residues involved in ethylene formation.

Results

We identified the open reading frame for the EFE homolog in Penicillium digitatum and also showed its capability of mediating ethylene production in yeast. The sequence of the EFE homologs from P.digitatum and P. syringae was compared to that of the non-functional EFE-homolog from Penicillium chrysogenum and ten amino acids were found to correlate with ethylene production. Several of these amino acid residues were found to be important for ethylene production via point mutations in P. syringae EFE. The EFE homolog from P. chrysogenum was engineered at 10 amino acid residues to mimic the P. syringae EFE, but this did not confer ethylene producing capability.Furthermore, we predicted the structure of EFE by homology to known structures of 2-oxoglutarate/Fe(II) dependent dioxygenases. Three of the amino acids correlating with ethylene production are located in the predicted 2-oxoglutarate binding domain. A protein domain specific for the EFE-class was shown to be essential for activity. Based on the structure and alanine substitutions, it is likely that amino acids (H189, D191 and H268) are responsible for binding the Fe(II) ligand.

Conclusion

We provide further insight into the structure and function of the ethylene forming (EFE) - subclass of 2-oxoglutarate/Fe(II) dependent dioxygenases. We conclude that residues in addition to the 10 identified positions implicated in ethylene production by sequence comparison, are important for determining ethylene formation. We also demonstrate the use of an alternative EFE gene. The data from this study will provide the basis for directed protein engineering to enhance the ethylene production capability and properties of EFE.

     

Redox reactivity of photogenerated osmium(II) complexes

Powerful reductants [Os(II)(NH(3))(5)L](2+) (L = OH(2), CH(3)CN) can be generated upon ultraviolet excitation of relatively inert [Os(II)(NH(3))(5)(N(2))](2+) in aqueous and acetonitrile solutions. Reactions of photogenerated Os(II) complexes with methyl viologen to form methyl viologen radical cation and [Os(III)(NH(3))(5)L](3+) were monitored by transient absorption spectroscopy. Rate constants range from 4.9 × 10(4) M(-1) s(-1) in acetonitrile solution to 3.2 × 10(7) (pH 3) and 2.5 × 10(8) M(-1) s(-1) (pH 12) in aqueous media. Photogeneration of five-coordinate Os(II) complexes opens the way for mechanistic investigations of activation/reduction of CO(2) and other relatively inert molecules.     

Structure and reactivity of homoleptic samarium(II) and thulium(II) phospholyl complexes

Potassium 2,5-di-tert-butyl-3,4-dimethylphospholide K(dtp) (9) was synthesised in 45 % yield from commercially available starting materials by using zirconacyclopentadiene chemistry. Reaction of the K salt of this bulky anion and of the previously described potassium 2,5-bis(trimethylsilyl)-3,4-dimethylphospholide K(dsp) (8) with SmI(2) in diethyl ether afforded the homoleptic samarium(II) complexes 7 and 6, respectively, whose solid-state structures, [[Sm(dtp)(2)](2)] (7 a) and [[Sm(dsp)(2)](2)] (6 a), are dimeric owing to coordination of the phosphorus lone pairs to samarium, as shown by X-ray crystallography. Reaction of 8 with TmI(2) in diethyl ether afforded [Tm(dsp)(2)(Et(2)O)], which could not be desolvated without decomposition. In contrast, the coordinated ether group of the solvate [Tm(dtp)(2)(Et(2)O)], obtained from 9 and TmI(2), could easily be removed by evaporation of the solvent and extraction with pentane at room temperature, and the monomer [Tm(dtp)(2)] (5) could be isolated and was characterised by X-ray crystallography. Presumably, steric crowding in 5 is too high for dimerisation to occur. Compound 5, the first Tm(II) homoleptic sandwich complex, is remarkably stable at room temperature in solution and did not noticeably react with nitrogen, in sharp contrast with other thulium(II) species. As expected, 5, 6 and 7 all reacted with azobenzene to give the trivalent complexes [Tm(dtp)(2)(N(2)Ph(2))] (13), [Sm(dsp)(2)(N(2)Ph(2))], (14) and [Sm(dtp)(2)(N(2)Ph(2))] (15), respectively; 13 and 14 were characterised by X-ray crystallography. Complex 5 immediately reacted with triphenylphosphane sulfide at room temperature to give [[Tm(dtp)(2)](2)(mu-S)] (16), which was characterised by X-ray crystallography, whereas samarium(II) complexes 6 and 7 did not noticeably react with Ph(3)PS over 24 h under the same conditions.     

Generation and reactivity of bis(2,6-diethylphenyl)germanium(II)

The title intermediate ($\text{3a}$) is produced on photolysis of hexakis(2,6-diethyl-phenyl)cyclotrigermane ($\text{1}$) or bis(2,6-diethylphenyl)bis(trimethylsilyl)germane ($\text{4}$) as evidenced by trapping experiments, and thermally dimerizes to tetrakis(2,6-diethyl-phenyl)digermene ($\text{2a}$). Diarylgermylenes such as $\text{3a}$ do not form stable triethylamine adducts (e.g. $\text{5a}$) as has been previously reported.     

Synthesis and reactivity of mono(amidinate) organoiron(II) complexes

The mono(amidinate) iron(ii) ferrate complex [{PhC(NAr)(2)}FeCl(micro-Cl)Li(THF)(3)] (1, Ar = 2,6-iPr(2)C(6)H(3)) was prepared and was found to undergo ligand redistribution in non-coordinating solvents to give the homoleptic [{PhC(NAr)(2)}(2)Fe] (2) as the only isolable product. Reaction of with alkylating agents also induces this redistribution, but the presence of pyridine allows isolation of the four-coordinate 14 VE monoalkyl complex [{PhC(NAr)(2)}FeCH(2)SiMe(3)(py)] (4). Generation of the 12 VE alkyl via pyridine abstraction from 4 by B(C(6)F(5))(3) again induced ligand redistribution. Attempts to trap a 12 VE alkyl species with CO led to the isolation of a dimeric Fe(0)-Li-ferrate complex (3) with a carbamoyl ligand, derived from CO insertion into the iron-amidinate bond.     

Nitric oxide reactivity of fluorophore coordinated carboxylate-bridged diiron(II) and dicobalt(II) complexes

The synthesis, structural characterization, and NO reactivity of carboxylate-bridged dimetallic complexes were investigated. The diiron(II) complex [Fe(2)(mu-O(2)CAr(Tol))(4)(Ds-pip)(2)] (1), where O(2)CAr(Tol) = 2,6-di(p-tolyl)benzoate and Ds-pip = dansyl-piperazine, was prepared and determined by X-ray crystallography to have a paddlewheel geometry. This complex reacts with NO within 1 min with a concomitant 4-fold increase in fluorescence emission intensity ascribed to displacement of Ds-pip. Although the diiron complex reacts with NO, as revealed by infrared spectroscopic studies, its sensitivity to dioxygen renders it unsuitable as an atmospheric NO sensor. The air-stable dicobalt(II) analogue was also synthesized and its reactivity investigated. In solution, the dicobalt(II) complex exists as an equilibrium between paddlewheel [Co(2)(mu-O(2)CAr(Tol))(4)(Ds-pip)(2)] (2) and windmill [Co(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(Ds-pip)(2)] (3) geometric isomers. Conditions for crystallizing pure samples of each of these isomers are described. Reaction of 2 with excess NO proceeds by reductive nitrosylation giving [Co(mu-O(2)CAr(Tol))(2)(NO)(4)] (5), which is accompanied by release of the Ds-pip fluorophore that is N-nitrosated in the process. This reaction affords an overall 9.6-fold increase in fluorescence emission intensity, further demonstrating the potential utility of ligand dissociation as a strategy for designing fluorescence-based sensors to detect nitric oxide in a variety of contexts.     

Oxygen reactivity of a nickel(II)-polyoximate complex

The ligand tris(2-hydroxyiminopropyl)amine (Ox(3)H(3)) binds to nickel(II) in multiple protonation states. In the neutral state, the X-ray crystal structure of the monomeric complex [Ni(Ox(3)H(3))(NO(3))(H(2)O)](NO(3)).(H(2)O), 1, has six-coordinate pseudo-octahedral geometry, with binding of the amine and three oxime nitrogens, a nitrate, and a water. In the mono-deprotonated form, the X-ray crystal structure shows a dimer, [Ni(Ox(3)H(2))(CH(3)CN)](2)(ClO(4))(2), 2, which has bridging oximate groups and a Ni-Ni distance of 3.575 A. The fully deprotonated complex, 3, shows significantly low Ni(II) oxidation potentials at -390 and +165 mV (versus Fc(+)/Fc). Complex 3 shows reactivity when exposed to O(2), consuming multiple O(2) equivalents and turning from the purple 3 to a dark brown complex, 4. Complex 4 has an EPR spectrum consistent with Ni(III), but spin quantitation accounts for only about 10% of the total Ni, consistent with turnover of the Ni oxidation states. This Ni(II)/O(2) system oxidizes triphenylphosphine to its oxide, with incorporation of the isotopic label from O(2).     

Low temperature activation and reactivity of CO2 over a Cr(II)-based heterogeneous catalyst: a spectroscopic study

A new heterogeneous catalyst for CO(2) activation was identified in the Cr(II)/SiO(2) Phillips catalyst, one of the most important catalysts used industrially for olefin polymerization. Interestingly, it was found that Cr(II)/SiO(2) strongly activates CO(2) already at room temperature, making it available for chemicals synthesis. A preliminary attempt in this direction was done by following the reaction of CO(2) with ethylene oxide at room temperature by means of FT-IR spectroscopy, which showed the formation of ethylene carbonate. Besides non-reductive CO(2) activation, Cr(II)/SiO(2) showed good performances in catalytic reduction of CO(2) to CO, when heated under mild conditions or irradiated with UV-Vis light. Both, in situ FT-IR and UV-Vis spectroscopy, were applied to highlight the redox process occurring at the Cr centres. These results open interesting perspectives to be developed in the field of CO(2) chemical fixation.     

Tuning the reactivity of osmium(II) and ruthenium(II) arene complexes under physiological conditions

The Os(II) arene ethylenediamine (en) complexes [(eta(6)-biphenyl)Os(en)Cl][Z], Z = BPh(4) (4) and BF(4) (5), are inactive toward A2780 ovarian cancer cells despite 4 being isostructural with an active Ru(II) analogue, 4R. Hydrolysis of 5 occurred 40 times more slowly than 4R. The aqua adduct 5A has a low pK(a) (6.3) compared to that of [(eta(6)-biphenyl)Ru(en)(OH(2))](2+) (7.7) and is therefore largely in the hydroxo form at physiological pH. The rate and extent of reaction of 5 with 9-ethylguanine were also less than those of 4R. We replaced the neutral en ligand by anionic acetylacetonate (acac). The complexes [(eta(6)-arene)Os(acac)Cl], arene = biphenyl (6), benzene (7), and p-cymene (8), adopt piano-stool structures similar to those of the Ru(II) analogues and form weak dimers through intermolecular (arene)C-H...O(acac) H-bonds. Remarkably, these Os(II) acac complexes undergo rapid hydrolysis to produce not only the aqua adduct, [(eta(6)-arene)Os(acac)(OH(2))](+), but also the hydroxo-bridged dimer, [(eta(6)-arene)Os(mu(2)-OH)(3)Os(eta(6)-arene)](+). The pK(a) values for the aqua adducts 6A, 7A, and 8A (7.1, 7.3, and 7.6, respectively) are lower than that for [(eta(6)-p-cymene)Ru(acac)(OH(2))](+) (9.4). Complex 8A rapidly forms adducts with 9-ethylguanine and adenosine, but not with cytidine or thymidine. Despite their reactivity toward nucleobases, complexes 6-8 were inactive toward A549 lung cancer cells. This is attributable to rapid hydrolysis and formation of unreactive hydroxo-bridged dimers which, surprisingly, were the only species present in aqueous solution at biologically relevant concentrations. Hence, the choice of chelating ligand in Os(II) (and Ru(II)) arene complexes can have a dramatic effect on hydrolysis behavior and nucleobase binding and provides a means of tuning the reactivity and the potential for discovery of anticancer complexes.