Oxidizing intermediates in cytochrome P450 model reactions

Oxoiron(IV) porphyrin -cation radicals have been considered as the sole reactive species in the catalytic oxidation of organic substrates by cytochromes P450 and their iron porphyrin models over the past two decades. Recent studies from several laboratories, however, have provided experimental evidence that multiple oxidizing species are involved in the oxygen transfer reactions and that the mechanism of oxygen transfer is much more complex than initially believed. In this Commentary, reactive intermediates that have been shown or proposed to be involved in iron porphyrin complex-catalyzed oxidation reactions are reviewed. Particularly, the current controversy on the oxoiron(IV) porphyrin -cation radical as a sole reactive species versus the involvement of multiple oxidizing species in oxygen transfer reactions is discussed.Abbreviations F₅PhIO pentafluoroiodosylbenzene - m-CPBA m-chloroperbenzoic acid - OEP dianion of octaethylporphyrin - PhIO iodosylbenzene - PPAA peroxyphenylacetic acid - TDCPP dianion of meso-tetrakis(2,6-dichlorophenyl)porphyrin - TMP dianion of meso-tetramesitylporphyrin - TPFPP dianion of meso-tetrakis(pentafluorophenyl)porphyrin - TPP dianion of meso-tetraphenylporphyrin - TTPPP dianion of meso-tetrakis(2,4,6-triphenylphenyl)porphyrin     

Phenotypic effects in Xenopus and zebrafish suggest that one-eyed pinhead functions as antagonist of BMP signalling

Zebrafish one-eyed pinhead (oep) is essential for embryonic axis and dorsal midline formation by promoting Nodal signalling and is thought to act as a permissive factor. Here we describe that oep elicits profound phenotypic effects when overexpressed in Xenopus and zebrafish. In Xenopus, wild-type oep inhibits mesoderm induction, disrupts axis formation and neuralizes animal caps. A secreted Oep dorsoanteriorizes and neuralizes Xenopus embryos indicative of BMP inhibition. In zebrafish, misexpression of smad1 in oep mutant embryos also reveals an interaction of oep with BMP signalling. Furthermore, the phenotypic effect of nodal overexpression can be rescued by coexpression of oep both in Xenopus and zebrafish. Taken together, our results support an interaction between oep and nodal but they suggest also (1) that the role of oep in Nodal signalling may include negative as well as positive regulation, (2) that oep is able to function in an active fashion and (3) that oep exerts a regulatory effect on the BMP signalling pathway.     

Reduction of Fe(III) porphyrin hydroxides by heterocyclic aromatic amines

The reduction of iron(III) porphyrin hydroxides by the heterocyclic aromatic amines, pyridine, 1-methylimidazole and derivatives, occurs in toluene to give the bisamine iron(II) porphyrin complexes. The reaction has not been fully characterized but is found to proceed through a different mechanism from that reported for the similar reductions by 1° and 2° amines in the absence of hydroxide ion. Preliminary data indicate that the first step in the reduction is formation of the bisamine Fe(III) porphyrin complex from the hydroxide. Nucleophilic attack by hydroxide ion on the aromatic ring of an axially ligated pyridine or methylimidazole of the Fe(III) complex followed by homolytic cleavage of the FeN bond is proposed.     

Inhibition of lipoxygenase (LOX) and anticancer activity caused by gold(I) mixed ligands complexes of triphenylphosphine and thioamides

Four mixed ligand gold(I) complexes with the thioamides 2-mercapto-thiazolidine (mtzdH), 2-mercapto-benzothiazole (mbztH) and 5-chloro-2-mercapto-benzothiazole (ClmbztH) and triphenylphosphine (tpp) of formulae [Au(tpp)Cl] (1) [Au(tpp)(mtzd)] (2), [Au(tpp)(mbzt)] (3) and [Au(tpp)(Clmbzt)] (4), already known, were used to study their mechanism of inhibition activity towards the catalytic oxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX), kinetically and theoretically. The results are compared to those of cisplatin. In addition, the anticancer cell screening results against leimyosarcoma (LMS) cells have shown that 2-4 complexes were more active than cisplatin. The uptake of complexes in LMS cells were also studied with electrospray ionisation mass spectrometry spectroscopy.     

Characterization of tpp1(+) as encoding a main trehalose-6P phosphatase in the fission yeast Schizosaccharomyces pombe

We have characterized an open reading frame of 2,454 bp on chromosome I of Schizosaccharomyces pombe as the gene encoding trehalose-6P phosphatase (tpp1(+)). Disruption of tpp1(+) caused in vivo accumulation of trehalose-6P upon heat shock and prevented cell growth at 37 to 40 degrees C. Accumulation of trehalose-6P in cells bearing a chromosomal disruption of the tpp1(+) gene and containing a plasmid with tpp1(+) under the control of the thiamine-repressible promotor correlated with tpp1(+) repression. The level of tpp1(+) mRNA rose upon heat shock, osmostress, or oxidative stress and was negatively controlled by cyclic AMP-dependent protein kinase activity. Expression of tpp1(+) during oxidative or osmotic stress, but not during heat shock, was under positive control by the wis1-sty1 (equivalent to phh1 and spc1) mitogen-activated protein kinase pathway. Analysis of Tpp1 protein levels suggests that the synthesis of trehalose-6P phosphatase may also be subjected to translational or posttranslational control.     

Formation of mu-oxo dimer by the reaction of (meso-tetraphenylporphyrinato)iron(III) with various imidazoles

Reactions between (meso-tetraphenylporphyrinato)iron(III) perchlorate [Fe(tpp)]ClO4 and various imidazoles have been examined in CD2Cl2 solutions. 1H NMR analysis revealed the formation of three kinds of complex; mu-oxo dimer, mono-imidazole adduct, and bis-imidazole adduct. The product ratios changed to a great extent depending on the amount and nature of imidazoles. In general, addition of less than 1.0 equiv of imidazole relative to [Fe(tpp)]ClO4 led to the formation of both mu-oxo dimer and mono-imidazole adduct. However, by the addition of excess amount of imidazole, either the mu-oxo dimer or bis-imidazole adduct was formed exclusively depending on the bulkiness of the imidazole used. In the case of bulky imidazole such as 2-methylbenzimidazole or 2-isopropyl-1-methylimidazole, the mu-oxo dimer was formed quantitatively. In the case of less bulky imidazole such as parent imidazole or 1-methylimidazole, bis-imidazole adduct became the sole product. The results have been explained in terms of the difference in steric interactions between the axial ligands and porphyrin core; the severe steric repulsion prohibits the formation of bis-adduct in the case of bulky imidazoles. As a result, bulky imidazoles prefer to behave as a base; they abstract a proton from coordinated water, and lead to the formation of mu-oxo dimer. Thus, the role of bulky imidazoles in these reactions has some relevance to that of distal histidine in hemoglobin and peroxidase.     

Correlation of macrocycle distortion with oxidation potentials of iron(III) porphyrins: molecule structure of the sterically crowded chloro-iron(III) 7,8,17,18-tetrabromo-5,10,15,20-tetraphenylporphyrin

The chloro-iron(III) complex of the tetraphenylporphyrin tetrabrominated at the antipodal β-pyrrole positions [(7,8,17,18-tetrabromo-5,10,15,20-tetraphenyl)porphyrin] has been synthesized and characterized by spectroscopy and X-ray crystallography. The iron atom is bonded to the chloride ion and the four pyrrole nitrogens. The Fe-Cl bond distance is 2.209(4) Å, and the mean value of the two opposite Fe-N_p lengths at the brominated pyrrole rings is 2.079(8) Å, whereas the mean value of the two opposite Fe-N_p′ bond distances at the non-brominated pyrrole rings is 2.041(8) Å. The X-ray structure determination and the analysis of the UV-Vis spectra obtained in solution and on thin films indicate that |FeCl(tpp-Br₄)| (1) is principally saddle-shaped in the solid state and in solution. Variable-temperature (195–325 K) ¹H NMR spectroscopy confirms the high-spin state (S=5/2) of the iron(III) center and indicates that the saddle-shaped conformation of 1 is maintained in solution. EPR spectra obtained in frozen CH₂Cl₂ solution and in the solid state show a rhombic symmetry with g values of 6.25, 5.70 and 1.99. Kadish et al. have shown that the one-electron oxidation potential of 1 increases only by 0.06?V relative to that of the non-brominated complex |FeCl(tpp)|. The present study indicates that the increase of the first oxidation potential of 1 is related to the non-planar distortion of the porphyrin. Relative to the unbrominated derivative |FeCl(tpp)|, this distortion destabilizes the π system of the macrocycle and thus compensates for the effects of the four electron-withdrawing bromine substituents.     

Studies on potential use of tin(IV) porphyrin in a role of proteins’ label

We present an electrochemical and optical characterization of 5,10,15,20-tetraphenylporphyrin tin(IV) dichloride (Sn–tpp) in terms of its potential use as a hybrid proteins’ label. Our research comprised Sn–tpp and Sn–tpp in the presence of model proteins selected as to mimic a receptor or surface blocking agents: bovine serum albumin, ovalbumin, and immunoglobulin G. In the course of the study, we determined optimal conditions for analysis by means of differential pulse voltammetry, ultraviolet–visible spectrophotometry, and spectrofluorimetry. In electrochemical detection, the influence of the working electrode, solvent, and supporting electrolyte was examined. Displacements of the received signals along the potential axis (a shift of the potential) and changes in signal intensities due to the addition of proteins were observed and analyzed. Simultaneously, the suitability of Sn–tpp as a label in optical detection mode was assessed by using spectroscopic techniques. The obtained results prove Sn–tpp to be applicable in dual and triple detection systems. Such an approach will improve the reliability of the analysis and, at the same time, will allow for widening the range of the linear response with some overlapping ranges of concentrations.     

Polymerization of horseradish peroxidase in the presence of inorganic adsorbents

Efficiencies of binding between horseradish peroxidase (HRP) and its polymers (HRPp) with inorganic adsorbents (precipitated and coprecipitated) were studied. In aqueous solutions, HRP efficiently adsorbed to aluminum oxide and the coprecipitated sorbent (composed of calcium orthophosphate, magnesium hydroxide, and aluminum hydroxide). HRP readily bound to zinc hydroxide but not to aluminum hydroxide in 25.0 mM bicarbonate buffer (pH 9.0). Several variants of HRP polymerization and HRPp modification with diamines in the presence of Al2O3 and Zn(OH)2 were compared. Synthesis of HRPp according to the scheme comprising HRP activation in solution followed by its polymerization in the presence of Zn(OH)2 appeared the most efficient. HRP and HRPp bound to Zn(OH)2 displayed a high catalytic activity in the presence of high H2O2 concentrations.     

Mosaic analysis with oep mutant reveals a repressive interaction between floor-plate and non-floor-plate mutant cells in the zebrafish neural tube

The floor plate is located at the ventral midline of the neural tube in vertebrates. Floor-plate development is severely impaired in zebrafish one-eyed pinhead (oep) mutants. oep encodes a membrane-bound protein with an epiblast growth factor (EGF) motif and functions autonomously in floor-plate precursors. To understand the cell behavior and cell-cell interaction during floor-plate development, the distribution and gene expression of wild-type and oep mutant cells in genetic mosaics were examined. When mutant shield cells were transplanted into a wild-type host, an ectopic neural tube with a floor plate was induced. However, the floor plate of the secondary axis was consistently devoid of mutant cells while its notochord was composed entirely of mutant cells. This indicates that oep shield cells adopt only a notochord fate in a wild-type environment. In reciprocal transplants (wild to oep), however, grafted shield cells frequently contributed to part of the floor-plate region of the secondary neural tube and expressed floor-plate markers. Careful examination of serial sections revealed that a mutant neural cell, when located next to the wild-type cells at the ventral midline, inhibited floor-plate differentiation of the adjacent wild-type cells. This inhibition was effective over an area only one- or two-cells wide along the anteroposterior axis. As the cells located at the ventral midline of the oep neural tube are thought to possess a neural character, similar to those located on either side of the floor plate in a wild-type embryo, this inhibition may play an important role during normal development in restricting the floor-plate region into the ventral-most midline by antagonizing homeogenetic signals from the floor-plate cells.     

Inhibition of lipoxygenase (LOX) and anticancer activity caused by gold(I) mixed ligands complexes of triphenylphosphine and thioamides

Four mixed ligand gold(I) complexes with the thioamides 2-mercapto-thiazolidine (mtzdH), 2-mercapto-benzothiazole (mbztH) and 5-chloro-2-mercapto-benzothiazole (ClmbztH) and triphenylphosphine (tpp) of formulae [Au(tpp)Cl] (1) [Au(tpp)(mtzd)] (2), [Au(tpp)(mbzt)] (3) and [Au(tpp)(Clmbzt)] (4), already known, were used to study their mechanism of inhibition activity towards the catalytic oxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX), kinetically and theoretically. The results are compared to those of cisplatin. In addition, the anticancer cell screening results against leimyosarcoma (LMS) cells have shown that 2-4 complexes were more active than cisplatin. The uptake of complexes in LMS cells were also studied with electrospray ionisation mass spectrometry spectroscopy.     

Substantial non-electrostatic forces are needed to induce allosteric disruption of thrombin’s active site through exosite 2

Sulfated β-O4 lignin (SbO4L), a non-saccharide glycosaminoglycan mimetic, was recently disclosed as a novel exosite 2-directed thrombin inhibitor with the capability of mimicking sulfated tyrosine sequences of glycoprotein Ibα resulting in dual anticoagulant and antiplatelet activities. SbO4L engages essentially the same residues of exosite 2 as heparin and yet induces allosteric inhibition. Fluorescence spectroscopic studies indicate that SbO4L reduces access of the active site to molecular probes and affinity studies at varying salt concentrations show nearly 6 ionic interactions, similar to heparin, but much higher non-ionic contribution. The results suggest that subtle increase in non-electrostatic forces arising from SbO4L’s aromatic scaffold appear to be critical for inducing allosteric dysfunction of thrombin’s active site.     

Durene-capped porphyrin complexes of iron(II). Binding of imidazoles, and spectroscopic trends within Fe(porp)B(L) species (B = imidazole base; L = RNC, CO, O2)

Solution equilibria are presented for in situ reactions of the type FeII(porp) + B----FeII(porp)B, where porp represents the dianion of some durene-capped porphyrins with variable length linking methylene straps, and B is 1-methyl, 1,2-dimethyl, or 1,5-dicyclohexylimidazole. Increasing distortion of the porphyrin skeleton has no effect on coordination of B at the unhindered side of the capped porphyrin. Increasing skeleton distortion gives rise to regular trends within the visible spectra of the free base porphyrins, the hemin (FeIII) chlorides, and six-coordinate FeII(porp)B(L) species (L = RNC, CO, O2), while the visible spectra of FeII(porp) and of FeII(porp)B species are independent of the nature of porp and B, which may be related to greater flexibility of the lower coordination number systems. v(CO) data for the FeII(porp)(CO)B species are discussed briefly.     

The EGF-CFC protein one-eyed pinhead is essential for nodal signaling

The zebrafish EGF-CFC gene one-eyed pinhead (oep) is required zygotically for the formation of the ventral neuroectoderm, endoderm, and prechordal plate. Here we report that embryos lacking both maternal and zygotic Oep activity are defective in germ layer formation, organizer development, and the positioning of the anterior-posterior axis. An identical phenotype is displayed by double mutants for the nodal-related genes squint and cyclops. Mutations in oep eliminate the response to Squint and Cyclops overexpression but are suppressed by expression of Activin and activated forms of the type I receptor ActRIB and Smad2. Expression of the murine EGF-CFC gene cripto rescues oep mutants. These results suggest a conserved role for EGF-CFC proteins as essential extracellular cofactors for Nodal signaling during vertebrate development.     

Monoporphyrinate neodymium (III) complexes stabilized by tripodal ligand: synthesis, characterization and luminescence

A series of monoporphyrinate neodymium (III) complexes stabilized by anionic tripodal ligand (cyclopentadienyl)tris(dimethylphosphito)cobaltate(I) were prepared and characterized by IR, ESI-HRMS, UV-Vis and X-ray diffraction studies. Structural analyses revealed that the Nd³⁺ ion was seven-coordinate, surrounded by four nitrogen atoms from the porphyrinate dianion and three oxygen atoms from the anionic tripodal ligand. Photoluminescence studies showed that the porphyrinate dianion acting as a sensitizer absorbed the light and transferred the energy to the Nd³⁺ center, which then allowed the metal ion to emit efficiently at 885 and 1071 nm. The luminescent intensity of the complexes increases when there are strong electronic donating groups on the porphyrin rings but decreases with increasing polarity of the solvents.     

Temporal and spatial expression of a thiolprotease gene during pea ovary senescence, and its regulation by gibberellin

Clones encoding a thiolprotease (tpp) have been isolated from a cDNA library of unpollinated, senescent pea ovaries and its pattern of expression during both ovary senescence and parthenocarpic development have been studied. The sequence of the tpp cDNA displays a high similarity with other plant and animal thiolproteases of the papain group. The homology is highest around the Cys-His of the active centre; a 109 amino acid sequence at the carboxy terminus was found to be homologous only to thiolproteases of plant origin; this part of the mRNA is also present in another pea mRNA that exhibits similar patterns of induction. tpp mRNA shows a temporal pattern of accumulation that precedes that observed for proteolytic activity. Such accumulation did not occur when ovaries were induced to grow parthenocarpically by gibberellic acid (GA) treatment; furthermore the initial low level of expression present in ovaries decreased after GA treatment, indicating that the gene is down-regulated by gibberellins. Spatially, tpp mRNA is localized mainly within the ovule and ovary vascular elements, and transiently within the endocarp of senescent ovaries. This pattern of expression precedes the development of the cytopathogenic effects observed as unpollinated ovaries undergo senescence.     

Osteoprotective effect of soybean and sesame oils in ovariectomized rats via estrogen-like mechanism

The purpose of the present study was to investigate the osteoprotective effects of soybean oil (SbO) and sesame oil (SO) in ovarictomized (OVX) rats. The results indicated that the OVX rats exhibited a significant decrease in Ca and P level in both serum and bone, the activities of the antioxidant enzymes SOD and CAT and the antioxidant biomarker GSH accompanied with a marked increase in the oxidative stress markers MDA and PC, the inflammatory indices (TNF-α, CRP levels, WBCs counts and ACP activity) in, both, bone and serum. Supplementating the diet of the OVX rats with SbO (15 % w/w) or SO (10 % w/w) for 2 months to resulted in modulation of the alterations in all tested parameters and succeeded to restore minerals, antioxidant enzymes, antioxidant biomarkers, oxidative stress markers, inflammatory indices, and WBCs counts. It could be concluded that the consumption of diets supplemented with SbO or SO might be useful for preventing bone loss caused by estrogen deficiency in ovariectomy status.     

Effect of alkaline pre-treatment on enzyme synergy for efficient hemicellulose hydrolysis in sugarcane bagasse

This aim of this study was to investigate the effect of ammonium hydroxide (NH₄OH) and sodium hydroxide (NaOH) pre-treatment on the digestibility of sugarcane bagasse (SCB) by hemicellulase action. It was found that pre-treatment of SCB with NH₄OH removed a larger percentage of the SCB lignin and effectively increased SCB digestibility 13.13 fold. The greatest amount of reducing sugar (1194.88 μmol/min) and largest degree of synergy (2.85) was obtained using a combination of two enzymes (25% ManA and 75% XynA) with NH₄OH pre-treated SCB. In this study, NH₄OH therefore appeared to be a more effective pre-treatment step for subsequent hydrolysis by hemicellulases.