Physicochemical properties of chitin-melanin and melanoprotein complexes from bee corpses

A technology for processing bee corpses and obtaining chitin-melanin and melanoprotein complexes has been developed. The obtained complexes of biopolymers were studied by the methods of absorption spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, thermogravimetry, and differential scanning calorimetry. The elemental composition of preparations was characterized. It was shown that the properties of the melanin-containing products of the processing of bee corpses are typical of chitin and melanin of animal origin. The results of EPR spectroscopy and thermal analysis are indicative of the diversity and structural complexity of the obtained products.     

Structures of manganese(II) complexes with ATP, ADP, and phosphocreatine in the reactive central complexes with creatine kinase: electron paramagnetic resonance studies with oxygen-17-labeled ligands

Coordination of Mn(II) to the phosphate groups of the substrates and products in the central complexes of the creatine kinase reaction mixture has been investigated by electron paramagnetic resonance (EPR) spectroscopy with regiospecifically 17O-labeled substrates. The EPR pattern for the equilibrium mixture is a superposition of spectra for the two central complexes, and this pattern differs from those observed for the ternary enzyme-Mn(II)-nucleotide complexes and from that for the dead-end complex enzyme-Mn(II)ADP-creatine. In order to identify those signals that are associated with each of the central complexes of the equilibrium mixture, spectra were obtained for a complex of enzyme, Mn(II)ATP, and a nonreactive analogue of creatine, 1-(carboxymethyl)-2-iminoimidazolidin-4-one, which is a newly synthesized competitive inhibitor. This inhibitor permits an unobstructed view of the EPR spectrum for Mn(II)ATP in the closed conformation of the active site. The EPR spectrum for this nonreactive complex with Mn(II)ATP matches one subset of signals in the spectrum for the equilibrium mixture, i.e., those due to the enzyme-Mn(II)-ATP-creatine complex. Chemical quenching of the samples followed by chromatographic assays for both ATP and ADP indicates that the enzyme-Mn(II)ADP-phosphocreatine and the enzyme-Mn(II)ATP-creatine complexes are present in a ratio of approximately 0.7 to 1. A similar value for the equilibrium constant for enzyme-bound substrates is obtained directly from the EPR spectrum for the equilibrium mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

EPR and O2.- scavenger activity: Cu(II)-peptide complexes as superoxide dismutase models.

Several copper(II) complexes with aminoacids and peptides are known to show superoxide dismutase (SOD)-like activity. EPR spectroscopy has proved to be a useful tool for studying the complex equilibria of the copper(II) ion and various ligands of biological importance in solution. In the present work, a variety of copper(II) complexes with di-, tri- and tetra-peptides containing only glycine residues (GG, GGG and GGGG) and others containing a histidyl residue in different positions (HGG, GHG, GGH and GGHG) have been investigated. EPR parameters obtained by extensive use of computer simulation of spectra lead to reliable spin Hamiltonian EPR parameters at both room temperature and in frozen solution. The molecular orbital coefficients computed from the anisotropic EPR data and the d-d electronic energies are used to characterize different arrangements of the complexes. Estimation of the scavenger activity of the complexes due to the particular environment created by the ligands around copper is discussed in the frame of the structure-activity relationship.     

Intracellular free iron in liver tissue and liver homogenate: studies with electron paramagnetic resonance on the formation of paramagnetic complexes with desferal and nitric oxide.

Treatment of intact liver and liver homogenate with sodium nitrite, or desferal, brings about the appearance of g = 2.03 and g = 4.3 electron paramagnetic resonance spectroscopy (EPR) signals, respectively. The g = 2.03 signal is conditioned by the formation of dinitrosyl complexes of Fe(II); the g = 4.3 signal is related to the appearance of paramagnetic desferal-Fe(III) complexes. Desferal and sodium nitrite were administered successively into liver homogenate, resulting in only a g = 4.3 EPR signal. And, vice versa, if desferal was administered after sodium nitrite, there appeared only the signal with g = 2.03. These data testify to the fact that one and the same endogenous free iron is included in both paramagnetic centers. The concentration of iron ions was measured in intact tissue according to the formation of dinitrosyl-iron complexes and desferal-iron complexes. It was 33.2 +/- 4.6 and 20.3 +/- 4.0 nmol/g of tissue weight, respectively. The data obtained testify to the fact that free endogenous iron is present in intact tissue. Possibilities of the EPR method for estimation of the content of intracellular free iron are discussed.     

Metallomic EPR spectroscopy

Based on explicit definitions of biomolecular EPR spectroscopy and of the metallome, this tutorial review positions EPR in the field of metallomics as a unique method to study native, integrated systems of metallobiomolecular coordination complexes subject to external stimuli. The specific techniques of whole-system bioEPR spectroscopy are described and their historic, recent, and anticipated applications are discussed.     

Heterogeneous Rieske proteins in the cytochrome b6f complex of Synechocystis PCC6803?

The completely sequenced genome of the cyanobacterium Synechocystis PCC6803 contains three open reading frames, petC1, petC2, and petC3, encoding putative Rieske iron-sulfur proteins. After heterologous overexpression, all three gene products have been characterized and shown to be Rieske proteins as typified by sequence analysis and EPR spectroscopy. Two of the overproduced proteins contained already incorporated iron-sulfur clusters, whereas the third one formed unstable aggregates, in which the FeS cluster had to be reconstituted after refolding of the denatured protein. Although EPR spectroscopy showed typical FeS signals for all Rieske proteins, an unusual low midpoint potential was revealed for PetC3 by EPR redox titration. Detailed characterization of Synechocystis membranes indicated that all three Rieske proteins are expressed under physiological conditions. Both for PetC1 and PetC3 the association with the thylakoid membrane was shown, and both could be identified, although in different amounts, in the isolated cytochrome b(6)f complex. The considerably lower redox potential determined for PetC3 indicates heterogeneous cytochrome b(6)f complexes in Synechocystis and suggests still to be established alternative electron transport routes.     

Evidence for variable metal-radical spin coupling in oxoferrylporphyrin cation radical complexes

Oxoferrylporphyrin cation radical complexes were generated by m-chloroperoxybenzoic acid oxidation of the chloro and trifluoromethanesulfonato complexes of tetramesitylporphyrinatoiron(III) [(TMP)Fe] and the trifluoromethanesulfonato complex of tetra(2,6-dichlorophenyl)porphyrinatoiron(III) [TPP(2,6-Cl)Fe]. Coupling between ferryl iron (S = 1) and porphyrin radical (S' = 1/2) spin systems was investigated by M?ssbauer and EPR spectroscopy. The oxoferrylporphyrin cation radical systems generated from the TMP complexes show strong ferromagnetic coupling. Analysis of the magnetic M?ssbauer spectra, using a spin Hamiltonian explicitly including a coupling tensor J, suggests an exchange-coupling constant J greater than 80 cm-1. The EPR spectra show non-zero rhombicity, the origin of which is discussed in terms of contributions from the usual zero-field effects of iron and from iron-radical spin-dipolar interaction. A consistent estimate of zero-field splitting parameter D approximately + 6 cm-1 was obtained by EPR and M?ssbauer measurements. EPR and M?ssbauer parameters are shown to be slightly dependent on solvent, but not on the axial ligand in the starting (TMP)Fe complex. In contrast to the TMP complex, the oxoferrylporphyrin cation radical system generated from [TPP(2,6-Cl)FeOSO2CF3] exhibits M?ssbauer and EPR spectra consistent with weak iron-porphyrin radical coupling of magnitude of J approximately 1 cm-1.     

Antioxidant capacity of mononitrosyl-iron-dithiocarbamate complexes: implications for NO trapping

Using EPR spectroscopy, we show that the water-soluble mononitrosyl iron complexes with N-methyl-D-glucamine dithiocarbamate (MNIC-MGD) ligands can easily react with superoxide and with peroxynitrite. The reaction with superoxide transforms the paramagnetic MNIC-MGD complex into an EPR silent complex with a reaction rate of 3 x 10(7) (M.s)(-1). Suppletion of ascorbate partially restores the complexes to their original paramagnetic state. We propose that the reaction of MNIC-MGD with either superoxide or peroxynitrite leads to identical EPR silent complexes. Our results have important implications for the technique of NO trapping in biosystems with Fe-dithiocarbamate complexes, where mononitrosyl-iron complexes (hydrophilic as well as hydrophobic) are formed as adducts in the trapping reaction. This principle is illustrated by NO trapping experiments on viable cultured endothelial cells. We find that MNIC-MGD acts as a very potent and water-soluble antioxidant with an efficiency exceeding most SOD mimics. Moreover, by accounting for the EPR silent fraction of iron complexes, the sensitivity of NO trapping can be enhanced considerably. The method was demonstrated for hydrophobic iron-dithiocarbamate complexes in endothelial cell cultures, where sensitivity for NO detection was enhanced by a factor of 5.     

Isolation of Chitin and Chitosan from Honeybees

A procedure of isolation of chitin, chitosan, and water-soluble low-molecular-weight chitosan from the corpses of bees has been developed. This procedure includes deproteinization of bee corpses, discoloration of the chitin–melanin complex, deacetylation, and enzymatic hydrolysis of chitosan.     

Electron paramagnetic resonance spectroscopy of thyroid peroxidase

Thyroid peroxidase was isolated from porcine thyroids by two methods. Limited trypsin proteolysis was employed to obtain a cleaved enzyme, and affinity chromatography was used to isolate intact thyroid peroxidase. Enzyme isolated by both methods was used in the examination of the heme site of native thyroid peroxidase and its complexes by EPR spectroscopy. Intact thyroid peroxidase showed a homogeneous high-spin EPR signal with axial symmetry, in contrast to the rhombic EPR signal of native lactoperoxidase. Reaction of cyanide or azide ion with native thyroid peroxidase resulted in the loss of the axial EPR signal within several hours. The EPR spectroscopy of the nitrosyl adduct of ferrous thyroid peroxidase exhibited a three-line hyperfine splitting pattern and indicated that the heme-ligand structure of thyroid peroxidase is significantly different from that of lactoperoxidase.     

EPR studies of copper(II) and cobalt(II) complexes of adriamycin

Cu2+ and Co2+ complexes of adriamycin (ADM) in aqueous solutions have been examined using EPR spectroscopy. An appreciable amount of Cu2+ and Co2+ complexes formed in the solutions were found to be in the EPR silent associated form, where the metal ions are antiferromagnetically coupled. The associated form of the Cu2+ complex may be neither a simple dimer nor coordination polymer but aggregates of a stacked type. Formation of a complex having Cu2+-ADM stoichiometry of 1:2 was observed for the solutions containing excess of ADM as an EPR observable species. The complex having Cu2+-ADM stoichiometry of 1:1 was not observed directly by EPR, but the presence of the complex is undeniable, especially at low pH range so far as large excessive ADM is not present. The Co2+ complex of ADM observed by EPR is in the high-spin (S = 3/2) state and may have a coordination structure of tetragonal symmetry. The EPR spectra of these complexes apparently show that the Cu2+ and Co2+ ions are bound at the carbonyl and phenolate oxygen in the 1,4-dihydroxyanthraquinone moiety and the amino nitrogen in the sugar part does not seem to participate in the coordination to the metal ions.     

EPR behavior of a soluble cardiac NADH dehydrogenase

A soluble, lipid free, NADH dehydrogenase isolated from cardiac muscle shows the characteristic iron-sulfide centers similar to those found in submitochondrial particles and mitochondria. The $\text{g}$-values obtained by low temperature EPR spectroscopy are 2.026, 1.943 and 1.927 for temperature “insensitive” center 1; 2.058 and 1.943 for center 2; and 2.111, 1.885 and 1.866 for centers 3 plus 4. The behavior of the EPR resonances suggests that iron may be coordinated not only with the labile-sulfide resulting in the iron-sulfide centers, but that iron is also liganded in complexes and spin-states as yet unidentified.     

Electron paramagnetic resonance dynamic signatures of TAR RNA-small molecule complexes provide insight into RNA structure and recognition

Electron paramagnetic resonance (EPR) spectroscopy was utilized to investigate the correlation between RNA structure and RNA internal dynamics in complexes of HIV-1 TAR RNA with small molecules. TAR RNAs containing single nitroxide spin-labels in the 2'-position of U23, U25, U38, or U40 were incubated with compounds known to inhibit TAR-Tat complex formation. The combined changes in nucleotide mobility at all four sites, as monitored by their EPR spectral width, yield a dynamic signature for each compound. The multicyclic dyes Hoechst 33258, DAPI, and berenil bind to TAR RNA in a similar manner and gave nearly identical signatures. Different signatures were obtained for the acridine derivative CGP 40336A and the aminoglycoside antibiotic neomycin, which bind to different regions of the RNA. The dynamic signature for guanidinoneomycin was remarkably similar to that obtained for argininamide and is evidence for guanidinoneomycin binding to the same site as arginine 52 of the Tat protein, rather than to the neomycin binding site. The data presented here show that the dynamic signatures provide strong insights into RNA structure and recognition and demonstrate the value of EPR spectroscopy for the investigation of small molecule binding to RNA.     

Thermodynamic and spectroscopic studies of Cu(II) and Ni(II) complexes with a new proline-threonine dipeptide ligand

The interactions between a new proline-threonine dipeptide ligand with two metallic cations were investigated in aqueous solution. The metallic cations studied were the copper(II) and the nickel(II), which are involved in many biological processes. The combination of potentiometry, UV-visible spectrophotometry, EPR, and mass spectrometry was used to determine the formation constants of the complexes and their structure in solution. The complexation sites were identified using electronic absorption and EPR spectroscopies. Copper complexes were obtained as square planar or square pyramidal mononuclear species, whereas nickel complexes were obtained as dinuclear species with an octahedral geometry.     

Inhibition of nitrogenase by nitrite and nitric oxide in Rhodopseudomonas sphaeroides f. sp. denitrificans

In cells of Rhodopseudomonas sphaeroides f. sp. denitrificans nitrite and nitric oxide, the products of denitrification, inhibit activity of nitrogenase enzyme.Ferredoxin-linked CO₂ fixation, with H₂ as a reductant, was also inhibited by nitrite and NO in denitrifying cells.EPR spectroscopy of cell preparations treated with NO showed that it reacts with non-haem iron-sulphur proteins to form iron-nitrosyl complexes. Nitrite also reacts with these iron-sulphur proteins, but the formation of ironnitrosyl complexes was dependent on the presence of dithionite. Since nitrite is reduced to NO by dithionite it is likely that nitrogenase and CO₂ fixation reactions are inhibited not only by nitrite itself, but also by nitric oxide.Abbreviation DPPH 1,1-diphenyl-2-picrylhydrazyl     

The binding of copper(II) and zinc(II) to oxidized glutathione

1H and 13C NMR studies of Zn(II) binding to oxidized glutathione (GSSG) in aqueous solution over the pH range 4-11 show that it forms a complex with a 1:1 Zn:GSSG stoichiometry. At pH values between 6 and 11 the metal ligands are the COO- and NH2 groups of the glutamate residues. Below pH 5 the glycine end of the molecule also binds to the metal ions. EPR and visible absorption spectra of Cu(II) GSSG solutions suggest that similar complexes are formed with Cu(II). The solid products obtained from these solutions are shown by analysis and EPR to be primarily binuclear with Cu2GSSG stoichiometry, although the structures depend on the pH and stoichiometry of the solution from which they were obtained.     

The DNA-bound orientation of Cu(II)-Xaa-Gly-His metallopeptides

The DNA-bound orientations of Cu(II) x Xaa-Gly-L-His metallopeptides (where Xaa is Gly, L-Lys or L-Arg) were investigated by DNA fiber EPR spectroscopy and molecular modeling. Observed and calculated EPR spectra indicated that the g// axes of 1:1 Cu(II) complexes of the tripeptides tilted about 50 degrees from the DNA fiber axis. These results suggest that the complexes are stereospecifically oriented in the DNA minor groove. Although the side chain of the N-terminal amino acid residue did not affect the orientation of the DNA-bound complexes, it contributed to their stability in the presence of DNA; the Cu(II) complex of Gly-Gly-L-His was found to dissociate to hydrated Cu(II) ion more extensively than the respective L-Lys-Gly-L-His and L-Arg-Gly-L-His complexes. The ionic interaction between the positively charged lysine or arginine residues and the negatively charged DNA phosphodiester backbone may result in the reduced dissociation of these complexes when bound to the DNA minor groove.     

Comparative EPR studies on the nitrite reductases from Escherichia coli and Wolinella succinogenes

Hexaheme nitrite reductases purified to homogeneity from Escherichia coli K-12 and Wolinella succinogenes were studied by low-temperature EPR spectroscopy. In their isolated states, the two enzymes revealed nearly identical EPR spectra when measured at 12 K. Both high-spin and low-spin ferric heme EPR resonances with g values of 9.7, 3.7, 2.9, 2.3 and 1.5 were observed. These signals disappeared upon reduction by dithionite. Reaction of reduced enzyme with nitrite resulted in the formation of ferrous heme-NO complexes with distinct EPR spectral characteristics. The heme-NO complexes formed with the two enzymes differed, however, in g values and line-shapes. When reacted with hydroxylamine, reduced enzymes also showed the formation of ferrous heme-NO complexes. These results suggested the involvement of an enzyme-bound NO intermediate during the six-electron reduction of nitrite to ammonia catalyzed by these two hexaheme nitrite reductases. Heme proteins that can either expose bound NO to reduction or release it are significant components of both assimilatory and dissimilatory metabolisms of nitrate. The different ferrous heme-NO complexes detected for the two enzymes indicated, nevertheless, their subtle variation in heme reactivity during the reduction reaction.     

Electron paramagnetic resonance and water proton relaxation rate studies of formyltetrahydrofolate synthetase-manganous ion complexes. Evidence for involvement of substrates in the promotion of a catalytically competent active site.

Conformational properties of the active site of formyltetrahydrofolate synthetase from Clostridium cylindrosorum have been examined by EPR spectroscopy and by solvent proton relaxation rate (PPR) studies of manganous complexes with the enzyme. Ternary enzyme-Mn-nucleotide complexes give EPR spectra which are very similar to those for the binary Mn-nucleotide complexes. However, upon addition of tetrahydrofolate to form the quaternary complexes, enzyme-MnADP-tetrahydrofolate and enzyme MnATP-tetrahydrofolate the EPR line shapes are changed substantially. Spectra for the quaternary complexes exhibit narrow line widths, and the splitting patterns are characteristic of a slightly asymmetric electronic environment for the bound Mn(II). Addition of formate to the ADP quatenary complex induces a further significant narrowing of the EPR line widths, although in the absence of tetrahydrofolate, formate does not influence the EPR spectrum for the enzyme-MnADP species. Both Pi and nitrate cause changes in the EPR patterns for the higher complexes of the enzyme which involve both ADP and tetrahydololate. However, the Pi effect is not influenced by the presence of formate whereas the characteristic effect of nitrate is potentiated only when formate is present. EPR sectra for the thernary complex with the beta, gamma-methylene analog of ATP App(CH2)p differ significantly from spectra for the binary App(CH)p complex is not influenced by further additions of tetrahydrofolate and of tetrahydorfolate and formate. The failure of spectra for the App(CH)p complex to respond to additions of the other substrates for the reaction is in marked contrast to the behavior found for the natural nucleotide substrates and is tentatively attributed to the lack of a protein-mediated interaction between the nucleotide and tetrahydrofolate binding sites in the analog complex. The frequency dependence of solvent PRR in the presence of the various complexes allows an estimate of the correlation times for electron-nuclear dipolar interaction and thereby the extent of hydration of the bound Mn(II) among the various complexes..     

Characterization and catechole oxidase activity of a family of copper complexes coordinated by tripodal pyrazole-based ligands

A family of tripodal pyrazole-based ligands has been synthesized by a condensation reaction between 1-hydroxypyrazoles and aminoalcohols. The diversity was introduced both on the substituents of the pyrazole ring and on the side chain. The corresponding copper(II) complexes have been prepared by reaction with CuCl₂ in tetrahydrofuran. They have been characterized by EPR, UV spectroscopy and cyclic voltammetry. The absence of the half-field splitting signals in EPR suggests that the complex exists in solution as mononuclear species. The influence of substituents and side chain of the tripodal ligand on the catecholase activity of the complexes was studied. The reaction rate depends on two factors. First, the presence of an oxygen atom in the third position of the side chain should be avoided to keep the effectiveness of the reaction. Second, the electronic and steric effects of substituents on the pyrazole ring strongly affect the catalytic activity of the complex. Thus, best results were obtained with complexes containing unsubstituted pyrazole based-ligands. Kinetic investigations with the best catalyst based on the Michaelis–Menten model show that the catalytic activity of the mononuclear complex is close to that of some dicopper complexes described in literature.