ESR an optical absorption studies on the copper(II) interaction with small peptides containing aromatic amino acids.

The interaction of Cu(II) with di- and tripeptides each containing phenylalanine, tryptophan or histidine in the amino acid chain has been investigated by means of electron spin resonance (ESR) and optical absorption spectroscopy. Cu(II) complexes of dipeptides and tripeptides exhibit different magnetic and optical parameters. Dipeptide complexes have larger gparallel-values and smaller A parallel values than tripeptide complexes. When compared to dipeptide complexes, the d-d band of the central metal ion is blue shifted for tripeptide complexes. There are no significant difference in the behavior of Cu(II) peptide complexes containing phenylalanine or tryptophan. Complexes of histidine containing peptides, however, show modified spectra caused by the participation of the imidazole nitrogen in the coordination to Cu(II). The imidazole nitrogen seems to coordinate in-plane with other coordinating atoms or in an axial position depending on the kind of peptide.     

Copper biosorption by chemically treated Micrococcus luteus cells

In order to clarify the binding states of copper in microbial cells, copper biosorption from aqueous systems using the chemically treated Micrococcus luteus IAM 1056 cells (hot water-treated, diluted NaOH-treated, chloroform–methanol-treated, and chloroform–methanol/concentrated KOH-treated cells) was examined. The intact cells of M. luteus adsorbed 527 mol of copper per g cells, and its copper adsorption was very rapid and was affected by the solution pH. The chloroform–methanol/concentrated KOH-treated cells showed higher copper biosorption capacity than the intact and the other chemically treated cells. The electron paramagnetic resonance (EPR) parameters, g and |A |, of Cu(II) ion in microbial cells indicate that Cu(II) ion in the intact and all the chemically treated cells have coordination environments with nitrogen and oxygen as donor atoms, being similar to those of type II proteins. The parameter g also indicated that the coupling between Cu(II) ion and the cell materials in the CHCl₃–MeOH/concentrated KOH-treated cells is rather more stable than those between Cu(II) ion and the cell materials in the other treated cells.     

Characterization of the copper(II) binding site in the pink copper binding protein CusF by electron paramagnetic resonance spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy has been used to structurally characterize the copper-binding site in CusF protein from Escherichia coli. The EPR spectra indicate a single type II copper center with parameters typical for nitrogen and oxygen ligands (A~200 G, g~2.186, g~2.051). The pulsed EPR data show that one of the ligands to Cu²⁺ is an imidazole ring of a histidine residue. The remote amino nitrogen of this imidazole ring is readily observed by electron spin-echo envelope modulation spectroscopy, while the imino nitrogen that is directly coordinated to the Cu²⁺ ion is observed by pulsed electron–nuclear double resonance (ENDOR). In addition, the ENDOR spectra reveal the presence of one more nitrogen ligand that was assigned to be a deprotonated peptide nitrogen. Apart from the two nitrogen ligands, it has been established that there are two nearby hydroxyl protons, although whether these belong to a single equatorial water ligand or two equatorial hydroxide ligands is not known.

Density Functional Theory Calculations of Molecular Hyperpolarizabilities

Recent density functional theory calculations of molecular hyperpolarizabilities are reviewed in order to try to assess the accuracy and reliability of DFT in the specified field by comparison with experiment and with ab initio HF and post-HF methods. In a table [1] (p. 157) containing results from the paper defining the compound electronic structure method Gaussian 2 [2], Foresman presents the relative accuracies of various 'model chemistries' for calculating thermochemical quantities. The Mean Absolute Deviation (MAD) from experiment, its Standard Deviation, and the largest positive and negative errors in the computed values, are the statistical means that allow the various models to be arranged in order of increasing MAD, and thus decreasing overall accuracy. These same statistical quantities, on a percent basis for size consistency and dimensionlessness, are used in this communication for quantifying the accuracies of various combinations of DFT functionals/basis sets/quantum chemical techniques/applied field strengths, in calculations of molecular mean dipole polarizabilities <>, first-order hyperpolarizabilities and second-order hyperpolarizabilities . The relative accuracies of DFT model chemistries are thus surveyed in three tables (including authors/references arranged in chronological order, compounds studied, and program codes employed), and can serve as guidelines for selecting optimal computational methodologies.     

Bohr-effect and pH-dependence of electron spin resonance spectra of a cobalt-substituted monomeric insect haemoglobin

The monomeric haemoglobin IV from Chironomus thummi thummi (CTT IV) exhibits an alkaline Bohr-effect and therefore it is an allosteric protein. By substitution of the haem iron for cobalt the O₂ half-saturation pressure, measured at 25 C, increases 250-fold. The Bohr-effect is not affected by the replacement of the central atom. The parameters of the Bohr-effect of cobalt CTT IV for 25 C are: inflection point of the Bohr-effect curve at pH 7.1, number of Bohr protons -log p_1/2 (O₂)/gDpH=0.36 mol H⁺/mol O₂ and amplitude of the Bohr-effect curve log p_1/2 (O₂)=0.84. The substitution of protoporphyrin for mesoporphyrin causes a 10 nm blue-shift of the visible absorption maxima in both, the native and the cobalt-substituted forms of CTT IV. Furthermore, the replacement of vinyl groups by ethyl groups at position 2 and 4 of the porphyrin system leads to an increase of O₂ affinities at 25 C which follows the order: proto < meso < deutero for iron and cobalt CTT IV, respectively. Again, the Bohr-effect is not affected by the replacement of protoporphyrin for mesoporphyrin or deuteroporphyrin. The electron spin resonance (ESR) spectra of both, deoxy cobalt proto- and deoxy cobalt meso-CTT IV, are independent of pH. The stronger electron-withdrawing effect by protoporphyrin is reflected by the decrease of the cobalt hyperfine constants coinciding with g=2.035 and by the low-field shift of g. The ESR spectra of oxy cobalt proto- and oxy cobalt meso-CTT IV are dependent of pH. The cobalt hyperfine constants coinciding with g=2.078 increase during transition from low to high pH. The pH-induced ESR spectral changes correlate with the alkaline Bohr-effect. Therefore, the two O₂ affinity states can be assigned to the low-pH and high-pH ESR spectral species. The low-pH form (low-affinity state) is characterized by a smaller, the high-pH form (high-affinity state) by a larger cobalt hyperfine constant in g. The correlation of the cobalt hyperfine constants of the oxy forms with the O₂ affinities is discussed for several monomeric haemoglobins. The Co-O-O bond angle in cobalt oxy CTT IV is characterized by an ozonoid type of binding geometry and varies little during the pH-induced conformation transition. Due to the lack of the distal histidine in CTT IV no additional interaction via hydrogen-bonding with dioxygen is possible; this is reflected by the cobalt hyperfine constants.     

Synthesis, characterization and antitumor studies of N-aroyl-N′-thioaroylhydrazines and their Co(II), Ni(II), Cu(II) and Zn(II) complexes

N-Salicyloyl-N-p-hydroxythiobenzohydrazide (H₂STPH) and N-benzoyl-N-thiobenzohydrazide (H₂BTBH) and their Co(II), Ni(II), Cu(II) and Zn(II) complexes were prepared and characterized by physicochemical studies. IR and NMR spectral studies imply dibasic tetradentate behaviour of the ligands bonding through `thiolato' sulfur, enolic oxygen and the two hydrazinic nitrogens in a polymeric fashion. The electronic spectra indicate [Ni(STPH)(H₂O)₂], [Co(STPH)(H₂O)₂] to be distorted octahedral while [Cu(BTBH)] has a square-planar geometry. In vitro antitumor results of the ligand and the complexes on P-815 (murine mastocytoma) and L-929 (murine fibroblast) indicate that these compounds show significant inhibition of ³H-thymidine and ³H-uridine incorporation in DNA and RNA, respectively, in these tumor cells at dose levels of 1, 2.5 and 5 g cm^–3. Antitumor studies suggest that [Cu(BTBH)] has significant dose dependent inhibitory effect on DNA synthesis. In vivo administration of [Cu(BTBH)] and [Ni(STPH)(H₂O)₂] resulted into prolongation of life span of Dalton's Lymphoma (DL) bearing mice.     

Cadmium-copper antagonism in the activation of periplasmic nitrous oxide reductase of copper-deficient cells fromPseudomonas stutzeri

Summary Copper-deficient cells ofPseudomonas stutzeri strain ZoBell synthesize catalytically inactive nitrous oxide (N₂O) reductase which is activated by added Cu(II) in the absence of de novo protein synthesis. The apparentK _m for the activation process is 0.13 M. Activation is temperature-dependent and is inhibited by Cd(II)(K _i 1.27 M) and less strongly by Zn(II), Ni(II), and Co(II). The same metal ions at 20 M have little or no effect on N₂O reduction of intact cells. Apo-N₂O reductase of transposon Tn5-inducednos ^– mutants with defective Cu-chromophore biosynthesis is not reactivated by Cu(II). N₂O reductase of Cu-sufficient and Cu-deficient wild type, and ofnos ^– mutants is localized in the periplasm, the latter providing the likely site of metal incorporation into the apoenzyme.     

Fluorescence polarization of stretched polytene chromosomes stained with acridine orange

The molecules of the fluorescent dye acridine orange (AO) bind to DNA in such a way that the absorption and emission dipoles lie on a plane perpendicular to the DNA axis. For this reason, definite fluorescence polarization should correspond to each mode of spatial DNA packing. A chromosome, considered as an axially symmetrical ensemble of DNA, was characterized by two experimental parameters, P and P , i.e., by polarizations of fluorescence excited by light polarized parallel and perpendicular to the symmetry axis. In view of the sequential order in the packing levels of DNA fiber in a chromosome, it was suggested that, under mechanical stretching, the highest level is disrupted first, then the others, in the order of their sequence.Isolated chromosomes of Chironomus thummi were stained with AO and stretched with needles of a micromanipulator. From the changes of P and P measured during stretching it was concluded the polytene chromosome bands have three, at least, DNA packing levels, tentatively described as 100 å fiber, 250 å coil and chromomere.     

A 5-methyltryptophan resistant rice mutant,MTR1, selected in tissue culture

Summary Cell lines resistant to tryptophan analogue 5-methyltryptophan (5MT) were selected in seed-derived calli of Oryza sativa L. var. Norin 8. Plants were regenerated (R₁ from one selected callus line (MTR1). In three out of the six R₁ plants, 5MT resistance was inherited in the R₂ and R₃ generations as a dominant nuclear mutation. Segregation ratios in the progeny of heterozygous plants were 11. Morphological and fertility variation seen in some of the R₂ plants were not correlated with 5-methyltryptophan resistance. Resistance in the MTR1 callus was due to the accumulation of high levels of free tryptophan (87-fold) that was associated with an increase in free phenylalanine content (9-fold). The leaves of resistant plants also contained elevated levels of free tryptophan and phenylalanine.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - 5MT D,L-5-methyltryptophan - phe phenylalanine - trp tryptophan - tyr tyrosine     

Redox behavior of copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b

The redox properties of the copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b were investigated. The ESR spectrum of the pMMO-containing membranes from M. trichosporium OB3b indicated a typical type II copper (II) signal (g = 2.24, A = 18.4 mT, g = 2.06, ₂= 0.84). By anaerobic addition of excess amounts of duroquinol, an optimum reductant of pMMO, the ESR spectra indicated that the copper cluster in membranes was reduced and successively oxidized by dioxygen, a substrate of pMMO. The result suggests that the copper is the active site of pMMO or an electron carrier. During the titration, the intensity of the type II copper signal decreased with decreasing potential and the multiple hyperfine structure at g = 2.06 appeared clearly. Although the copper signal did not change by treatment of the EDTA-treated membranes with duroquinol and dioxygen, the copper signal intensity decreased with decreasing potential in the redox titration. These results suggest that some redox mediators play a role as an electron carrier between the active site and a reductant, and the presence of at least two types of copper sites in pMMO- containing membranes. On the basis of the ESR spectra of the EDTA-treated membranes and the as-isolated membranes, it is concluded that one type of the copper sites functions as the active site of pMMO (A-site), and the other type of copper sites plays a role as an electron carrier (E-site)     

Oxidant types in copper–dioxygen chemistry: the ligand coordination defines the Cu<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript>-O<Subscript>2</Subscript> structure and subsequent reactivity

The considerable recent advances in copper–dioxygen coordination chemistry demonstrate the existence of a variety of dioxygen-derived Cu_n-O₂ complexes, forming a basis for discussion of alternate oxidant types in copper chemistry and biochemistry. Peroxo complexes may react as nucleophilic reagents, and several types of electrophilic mono- or dicopper (hydro)peroxides exist. Side-on peroxo-dicopper(II) species effect aromatic hydroxylations, including phenolic substrates, in model systems and in the enzyme tyrosinase. Bis--oxo-dicopper(III) entities are capable of hydrogen-atom abstraction reactions, or atom transfer to phosphines and sulfides. The scope and mechanisms of mononuclear Cu(II) superoxides or peroxides are yet to be established, but may be relevant to monooxygenases like peptidylglycine -hydroxylating monooxygenase.     

Copper-glutathione complexes under physiological conditions: structures in solution different from the solid state coordination

The physiologically important copper complexes of oxidized glutathione have been examined by electron spin resonance (ESR) spectroscopy in aqueous solution at neutral pH. Low temperature measurements show that the Cu(II) binding site in oxidized glutathione has the same ligand arrangement as in the copper complexes of S-methylglutathione, glutamine, glutamate and glycine. The site is composed of the amino nitrogens and the carboxyl oxygens of two -glutamyl residues; there is no interaction with amide nitrogens, the sulphur bond or the glycyl carboxyl groups. At high metal to ligand ratios a binuclear species exists, in which each Cu(II) binds only to one -glutamyl residue. The previously reported forbidden transition detected at g = 4 is due to non-specific aggregation and not to spin coupling of intramolecular sites. Liquid solution ESR spectra show the Cu(II)-glutathione complex has a lower mobility than the corresponding Cu(II)-S'-methylglutathione species. From the degree of spectral anisotropy the complex with glutathione is calculated to exist as a dimer. These results demonstrate that the physiologically relevant complex between copper and oxidized glutathione in solution is completely different from the known solid state structure determined by crystallography.     

Synthesis, Characterization and Antitumor Studies of Mn(II), Ni(II), Cu(II) and Zn(II) Complexes of N-Nicotinoyl-N′-o-Hydroxythiobenzhydrazide

A new ligand N-Nicotinoyl-N-o-hydroxythiobenzhydrazide (H₂Notbh) forms complexes [Mn(Notbh)(H₂O)], [M(Notbh)] [M=Ni(II) Cu(II) and Zn(II)] which were characterized by various physico-chemical techniques. All the metal complexes were observed to inhibit the growth of tumor in vitro, whereas, ligand did not. In vivo administration of these complexes resulted in prolongation of survival of tumor bearing mice. Tumor bearing mice administered with metal complexes showed reversal of tumor growth associated induction of apoptosis in lymphocytes. The paper discusses the possible mechanisms and therapeutic implication of the H₂Notbh and its metal complexes in tumor regression and tumor growth associated immunosuppression.     

Binding of [3H]muscimol to calf cerebrocortical synaptic membranes and the effects of sulphur-containing convulsant and non-convulsant compounds

Endogenous and xenobiotic sulphur-containing convulsant and non-convulsant compounds containing structural moieties of, or bearing a structural resemblance to, GABA and homocysteine were tested in binding studies for their potency in displacing the GABA-mimetic [³H]muscimol from specific, high-affinity sites (K _d=3.6 nM;B _max=3.94 pmol/mg protein) on freeze-thawed, Triton-treated calf-brain synaptic membranes. The xenobiotic convulsants, 4-mercaptobutyric acid (MBA), 3-mercaptopropionic acid (3-MPA) and 2-mercaptopropionic acid (2-MPA) were found to be two-site competitive inhibitors exhibiting apparent inhibition affinity constants (K _i ^app ) of 5000 M, 3750 M, and 4800 M, respectively; while homocysteic acid (K _i ^app =4800 M) was shown to be a one-site partial competitive inhibitor. Intermediary metabolites of methionine: S-adenosyl-l-homocysteine,l-cysteine, the convulsantl-homocysteine, and its non-convulsant disulphide oxidation product, homocystine, were found to be one-site partial competitive inhibitors exhibitingK _i ^app values of 5750 M, 8350 M, 5000 M, and 510 M, respectively. The endogenous anticonvulsant neuroeffector, taurine, and the tripeptide, reduced glutathione (GSH) were shown to be, respectively, one-site (K _i=20 M) and two-site (K _i ^app =4300 M) competitive inhibitors of [³H]muscimol binding. These findings are discussed with regard to a previously proposed mechanism for the convulsant action of homocysteine.     

Dynamic studies of a fibronectin type I module pair at three frequencies: Anisotropic modelling and direct determination of conformational exchange

Summary A detailed analysis of the ¹⁵N relaxation of a pair of modules from fibronectin is presented. The overall dimensions of the protein structure can be approximated by a cylinder with an axial ratio D/D of 1.9. T₁, T₂ and NOE data, collected at three ¹⁵M frequencies (50.6, 60.8 and 76 MHz), can be fitted satisfactorily to a Lipari-Szabo model, taking anisotropy into account. A method for analysing the exchange contribution to relaxation is presented. This contribution depends upon the predicted B _inf0 ^sup2 frequency dependence in the fast exchange limit of these exchange terms. Using this analysis, relatively slow conformational exchange contributions are detected around one of the disulphide bonds in the first module of the pair.     

Mesogenic and magnetic properties of nickel(II) complexes formed from 2′-(4-alkyloxyphenyl)malondialdehydes

The synthesis, characterization, thermal behavior and magnetic properties of a number of bis[2-(4-alkyloxyphenyl)malondialdehyde] nickel(II) complexes are reported. X-ray studies of the nickel(II) complexes were performed. It was found that the nickel(II) complexes show liquid-crystalline smectic A phases over a broad temperature range with low melting points. The temperature-dependent magnetic susceptibility measurements of the bis[2-(4-decyloxyphenyl)malondialdehyde] nickel(II) complex were carried out in the range of 4.2–480 K. The temperature dependent magnetic susceptibilities and magnetic moments of this compound (_eff=3.27 _B at 300 K) indicate that the nickel centers are octahedrally coordinated. Models for the molecular arrangement in the crystalline and liquid-crystalline phases are discussed on the basis of the magnetic data. In spite of the oxygen bridge between the nickel centers, no exchange interactions were found in the crystalline and liquid-crystalline phases.     

Production of carbon monoxide from aromatic amino acids by Morganella morganii

Morganella morganii produced CO when cultured in a medium containing casamino acids or peptone as the sole carbon source. Although the production of CO was distinctly enhanced by the addition of hemin to the medium, the amounts of CO produced in the absence of hemin were nearly proportional to the amounts of peptone added to the culture media. Examination of 20 amino acids for their ability to produce CO by resting cells revealed that phenylalanine, tyrosine, histidine and tryptophan were the sources of CO. Oxygen and hemin were necessary for CO production from the amino acids except tryptophan which produced CO in the absence of hemin. When cells were incubated for 4 h at 30° C in the mixture containing 40 mol tyrosine and 1 mol hemin, about 15 mol CO was produced; the activity of CO production was about 1.2 mol CO/h · mg cell nitrogen. Phenylpyruvic acid, p-hydroxyphenylpyruvic acid and imidazolepyruvic acid also produced CO in the presence of hemin, while indolepyruvic acid produced CO regardless of the presence or absence of hemin. The production of CO by the 2-oxo acids proceeded spontaneously and did not require the presence of M. morganii cells.     

Localization of tryptophan rich proteins and β-glycerophosphatase activity inCuscuta haustorial cells

Summary The DMAB-nitrite method for tryptophan containing proteins and-GPase activity has been studied in the cells of the cortex adjoining the haustonal cells ofCuscuta pentagona A strong positive reaction with DMAB-nitrite method points to a similarity between these cells and the animal ones secreting pepsidases. A-GPase activity has been also found in the parasite cells in contact with the host stem. A protein rich in tryptophan as well as the-GPase activity might to be involved in the lysis of the host tissues.     

Amino Acid Sequence, Spectral, Oxygen-Binding, and Autoxidation Properties of Indoleamine Dioxygenase-Like Myoglobin from the Gastropod Mollusc Turbo cornutus

Myoglobin was isolated from the radular muscle of the archaeogastropod mollusc Turbo cornutus (Turbinidae). This myoglobin is a monomer carrying one protoheme group; the molecular mass was estimated by SDS–PAGE to be about 40 kDa, 2.5 times larger than that of usual myoglobin. The cDNA-derived amino acid sequence of 375 residues was determined, of which 327 residues were identified directly by chemical sequencing of internal peptides. The amino acid sequence of Turbo myoglobin showed no significant homology with any other usual 16-kDa globins, but showed 36% identity with the myoglobin from Sulculus diversicolor (Haliotiidae) and 27% identity with human indoleamine 2,3-dioxygenase, a tryptophan-degrading enzyme containing heme. Thus, the Turbo myoglobin can be counted among the myoglobins which evolved from the same ancestor as that of indoleamine 2,3-dioxygenase. The absorbance ratio of to CT maximum (/CT) of Turbo metmyoglobin was 17.8, indicating that this myoglobin probably possesses a histidine residue near the sixth coordination position of heme iron. The Turbo myoglobin binds oxygen reversibly. Its oxygen equilibrium properties are similar to those of Sulculus myoglobin, giving P ₅₀ = 3.5 mm Hg at pH 7.4 and 20°C. The pH dependence of autoxidation of Turbo oxymyoglobin was quite different from that of mammalian myoglobin, suggesting a unique protein folding around the heme cavity of Turbo myoglobin. A kinetic analysis of autoxidation indicates that the amino acid residue with pK _a = 5.4 is involved in the reaction. The autoxidation reaction was enhanced markedly at pH 7.6, but not at pH 5.5 and 6.3 in the presence of tryptophan. We suggest that a noncatalytic binding site for tryptophan, in which several dissociation groups with pK _a 7.6 are involved, remains in Turbo myoglobin as a relic of molecular evolution.