EPR Spectroscopic Studies Research Articles

Effect of a water-miscible organic solvent on the kinetic and structural properties of trypsin

The effects of a water-miscible organic solvent (9:1 mix of DMF and DMSO) on the esterase and amidase kinetic parameters of trypsin have been investigated. The solvent selectively reduced the k cat for amidase activity relative to that for esterase activity. For both reaction types, K m increased sharply with increasing solvent concentration. Kinetic studies were complemented by fluorescent measurements of the enzyme's overall conformation and by EPR spectroscopy studies of the active-site microstructure. The decline in k cat for amidase activity began at solvent concentrations greater than 10%; this decrease correlated well with minor changes in the structure of the active site as indicated by EPR spectroscopy. In contrast, the esterase k cat showed no significant decrease throughout the solvent concentration range studied. The k cat values were based on the number of active sites as indicated by active site titration.

Visible, EPR and electron nuclear double‐resonance spectroscopic studies on the two metal‐binding sites of oxovanadium (IV)‐substituted D‐xylose isomerase

The two metal-binding sites of the D-xylose isomerase from Streptomyces rubiginosus were studied using VO2+ as a sensor for the ligand environment. Titration of the tetrameric enzyme with VO2+, followed by EPR spectroscopy and inhibition studies, show that the first four VO2+ equivalents occupy, in analogy to Co2+, Cd2+ and Pb2+, the binding site B. The visible absorption data and the EPR parameters indicate that a nitrogen ligand is involved in the ligand sphere of the high-affinity B site. The low-affinity A site could be studied selectively by blocking the B site with visible and EPR-silent Cd2+. The visible data and EPR parameters for this site are consistent with a ligand environment composed of oxygen donors without nitrogen ligation. The nitrogen coordination in the high-affinity site could be demonstrated by electron nuclear double-resonance (ENDOR) studies of the 4VO2+ enzyme, and was assigned to a histidine ligand. The 14N resonances are interpreted in terms of a quartet with a coupling value of 13.2 MHz. 1H-ENDOR coupling of 1.7 MHz, exchangeable in D2O, has been assigned to the N-H proton of the histidine. Additional proton ENDOR couplings, which are not exchangeable, are due to protons bound to the carbon atoms of the histidine. For the low-affinity binding site, a nitrogen coordination could be definitely excluded by the ENDOR measurements. Exchangeable 1H-ENDOR couplings observed in this sample were assigned to H2O ligands in the vicinity of VO2+. The results closely relate to what is known from X-ray structure. However, the relative affinities for the two binding sites seem not to be the same for different bivalent cations. In mixed metal samples with four VO2+ and four Co2+ equivalents, the VO2+ is distributed between both binding sites. Small changes in the complex geometry of the A site, indicated by different EPR features, seem to occur if the B site is occupied by Co2+ or by Cd2+.

Structural rearrangements in active and inactive forms of hydrogenase from Thiocapsa roseopersicina.

The electrophoretic behavior of Thiocapsa roseopersicina hydrogenase on sodium dodecyl sulfate gels demonstrates that the protein exists in two active forms, A1 and A2, which may be interconverted. Each of these forms has a characteristic electrophoretic mobility and differs in its sensitivity to O2. Form A1 is O2-labile and converts to A2 under O2. Form A2 is less sensitive to O2 and may be converted into A1 under H2 atmosphere. Both active forms are present in aerobically isolated samples. Because the proteins are still active on 15% sodium dodecyl sulfate gels, they are not completely denatured, and the apparent molecular masses do not necessarily represent the true molecular masses of the enzymes. A1 has an Rf = 0.19, corresponding to an apparent molecular mass of 90 kDa, and A2 has an Rf = 0.35, corresponding to an apparent molecular mass of 49 kDa. A sedimentation equilibrium centrifugation study of the active enzyme shows that the holoenzyme has a molecular mass of 98 kDa. Form A2 may be separated into two subunits of molecular mass of 64 kDa and 34 kDa, respectively. Thus, form A2 represents the holoenzyme with a true molecular mass of 98 kDa. Amino acid compositions and N-terminal amino acid sequences of the A2 protein and these subunits are consistent with a heterodimeric holoenzyme. The relationship between the conformational changes detected in this study and a three-state scheme proposed on the basis of EPR spectroscopic studies of the metal-containing cofactors present in the enzyme is also discussed.

ChemInform Abstract: Preparation, Characterization, and Structural Aspects of Transition‐Metal Complexes with 4,7,10‐Trioxa‐1,13‐dithia(13)(2,5)‐1,3,4‐thiadiazolophane.

AbstractThe macrocyclic title ligand (I) reacts with metal nitrates, CuBr2 and CdCl2 in absolute, refluxing MeOH to produce the complexes (II)‐(IV), which are characterized by EPR and FAB mass spectroscopy and electronic spectral studies.

Epr Spectroscopic Studies of Detection of a Carbon-Centered Free Radical During Acetylcholine-Induced and Endothelium-Dependent Relaxation of Guinea Pig Pulmonary Artery

Vascular smooth muscle relaxation by several vasodilators, including acetylcholine (Ach) and ATP, depends on the presence of intact endothelium. Ach is thought to activate muscarinic receptors on endothelium to release an endothelium-derived relaxing factor (EDRF) which brings about relaxation of smooth muscle. In order to assess the role of free radicals in the endothelium-dependent relaxation of blood vessel, we have studied the effect of a spin-trapping agent, phenyl t-butyl nitrone (PBN), on Ach-, ATP-, and sodium nitroprusside-induced relaxation of guinea pig pulmonary artery. Arterial strips were mounted in a 5-ml organ bath containing Krebs solution equilibrated with 95% O2 and 5% CO2 at 37 degrees C. After increasing vascular tone by a synthetic prostaglandin endoperoxide analog (50 ng/ml), the strips relaxed dose-dependently in response to Ach (5 x 10(-8) M), ATP (1.5 x 10(-6) M) or sodium nitroprusside (6 x 10(-9) M). Removal of the endothelium abolished the relaxation by Ach or ATP, but did not affect the relaxation by sodium nitroprusside. PBN inhibited Ach-induced relaxation of pulmonary artery dose-dependently, but had no effect on relaxations by ATP or sodium nitroprusside. PBN did not block radioligand binding to muscarinic cholinergic membrane receptors on both chick embryonic heart and guinea pig pulmonary artery endothelial cells indicating that it does not block the muscarinic receptors. Spin trapping in combination with electron paramagnetic resonance (EPR) spectral analysis revealed a carbon-centered radical with hyperfine splitting constants of aN = 16.0 G and aH beta = 3.85G in the lipid extracts of pulmonary artery (0.2-0.4 g) incubated with PBN (14 mM) and Ach (3 x 10(-6) M) for 20 min. No signal was detected when endothelium was removed. Our data suggest that the endothelium-dependent relaxation of pulmonary artery by Ach is associated with the generation of a free-radical and can be prevented by a spin-trapping agent. ATP, however, relaxes the arterial smooth muscle by a different mechanism.

The role of nickel and iron-sulfur centers in the bioproduction of hydrogen

Abstract

Catalytic properties and active‐site structural features of immobilized horse liver alcohol dehydrogenase

Alcohol dehydrogenase from horse liver was immobilized by covalent attachment to CNBr-Sepharose and by adsorption to octyl-Sepharose CL-4B, a hydrophobic analog of Sepharose. In each case, rate constants for the binding and release of coenzyme and for the oxidation of substrates were measured based on the concentration of accessible active-site zinc atoms determined by titration with a paramagnetic inhibitor. All rate constants were substantially reduced upon immobilization; however, the rate constant of immobilized enzyme for ethanol oxidation was independent of the immobilization method, whereas the rate constant for cyclohexanol oxidation was lower for enzyme immobilized to octyl-Sepharose. Consequently, the substrate specificity of the two immobilized enzyme samples differed by an order of magnitude. Moreover, EPR spectroscopy studies and computer graphic analyses of spin labels occupying three defined regions of the active-site domain indicated that the active-site conformation adjacent to the catalytic zinc atom was similar in the two samples while the conformation slightly further from the zinc atom was different. This result may explain why the two immobilized enzyme preparations exhibited the same rate constant toward a small substrate (ethanol) yet different rate constants toward a larger substrate (cyclohexanol), whose rate constant is expected to be sensitive to a larger portion of the active site.

Optical, EPR and Mössbauer spectroscopic studies on the NO derivatives of cytochrome cd1 from Thiobacillus denitrificans.

We have used optical, EPR and Mössbauer spectroscopies to study the formation of heme-NO complex upon the addition of nitrite to reduced cytochrome cd1 from Thiobacillus denitrificans. The reduced d1 heme binds NO under both alkaline and acidic conditions, but the binding of NO to the reduced c heme was strongly pH-dependent. The Mössbauer data showed unambiguously that at pH 7.6 the c heme does not complex NO, whereas at pH 5.8 approximately half of the reduced c heme binds NO. This observation was confirmed by EPR studies, which showed that the spin concentration of the heme-NO EPR signal increased from 2 spins/molecule at pH 8.0 to approximately 3 spins/molecule at pH 5.8. Optical absorption study also showed strong pH dependence in the binding of NO to the reduced c heme. We have also analyzed the Mössbauer spectra of the ferrous d1 heme-NO complex using a spin-Hamiltonian formalism. The magnetic hyperfine coupling tensor was found to be consistent with the unpaired electron residing on a sigma orbital.

Mössbauer and EPR spectroscopic study of the iron-EDTA system

Mössbauer and EPR spectroscopic studies of the iron(III)-EDTA system have shown that primarily the mode of preparation (the reaction of iron(III) with the ligand, or oxidation of the iron(II) complex) determines the iron(III)-iron(III) spin-spin interaction (dimer formation) in the complexes. Increases in the concentration and the pH also promote the dimerization process. The pH-dependence of the Mössbauer spectra of the iron(II)-EDTA complexes revealed the formation of a protonated complex, besides the parent complex, in acidic solution.

EPR and UV-visible spectroscopic studies of copper(II) and cobalt(II) complexes of hydroxyanthraquinones

Interactions of 1-hydroxyanthraquinone (1-HAQ) and 1,4- and 1,8-dihydroxyanthraquinone (1,4-DHAQ, 1,8-DHAQ) with Co 2+ and Cu 2+ have been examined by UV-visible and EPR spectroscopies. These hydroxyanthraquinones form high-spin complexes with Co 2+. It has been estimated that these Co 2+ complexes in dichloromethane and ethanol have a tetragonal coordination structure, while in dimethlyformamide (DMF) or in mixed solvents containing DMF the complex having a tetrahedral structure is simultaneously formed. 1-HAQ forms the ML and ML 2 type complexes with Cu 2+, their relative concentration depending on the ligand to metal mole ratio, R, in solution. 1,4-DHAQ forms also the ML 2 type complex with Cu 2+, but the ML-type mononuclear Cu 2+ complex was not directly detected by EPR, though 1,4-DHAQ was estimated to form a polymer-type complex with Cu 2+ having metal-ligand ratio of ~ 1:1. For the case of 1,8-DHAQ, the formation of S = 1 M 2L 2-type dinuclear complex was observed, in addition to the formation of ML- and ML 2-type complexes. The ML 2-type Cu 2+ complexes of 1,4- and 1,8-DHAQ were found to have two forms, the cis and trans configurations. These properties for metal complex formation of the hydroxyanthraquinones are compared with those of adriamycin, which has a similar anthraquinone structure in the molecule.

Structural investigations of semiconducting barium-vanadate glasses containing iron

Mossbauer and EPR spectroscopic studies are performed on (0.9-z)V2O5.ZBaO. 0.1Fe2O3 amorphous semiconductors. It is observed that the addition of BaO results in the formation of non-bridging oxygens. For low Z-values the formed non-bridging oxygens are attached to vanadium ions whereas for higher Z-values some of the oxygens attached to Fe3+ ions are also converted into non-bridging ones.

Optical and EPR spectroscopy studies on haem arginate, a new compound used for treatment of porphyria

A protohaem compound, used for treatment of porphyrias, has been studied to elucidate its state of aggregation. EPR and absorption spectroscopy measurements reveal that 38.3 mM protohaem, dissolved in 40% 1,2-propanediol/10% ethanol/water solution, also containing 153 mM arginine, is partly EPR silent. It exists as high molecular weight aggregates and probably also as μ-oxo-dimers. Dilution in the aqueous alcohol solution dissolves the aggregates first to oligomers and dimers, and finally to monomers ( K diss = 24 × 10 −6 M). When haem is diluted in 0.9% sodium chloride, a fully monomeric state is not reached even at 1 μM concentration. At 3.5 μM concentration, that used for infusion in patients, the haem is still totally aggregated.

Hyperfine interaction of polyacetylene intercalated with iron halide anions: 57Fe Mössbauer and EPR spectroscopy studies

57Fe Mössbauer spectroscopy and EPR spectroscopy were applied to study the chemical nature of the intercalant in polyacetylene oxidized (doped) chemically with FeCl 3 and FeBr 3. For the (CH) x/FeCl 3 system the Mossbauer lattice temperature ( θ M = 89 K) was estimated from the temperature dependence of the recoil-free fraction. The strong influence of the high Fe III state of the dopant is clearly evidenced through EPR spectra of both systems: while the chloride system shows a superposition of a narrow and a broad lines, the bromide one exhibits only a narrow line, whose intensity decreases with increasing dopant concentration.

On the environment and the rotational motion of amphiphilic flavins in artificial membrane vesicles as studied by electron paramagnetic resonance spectroscopy

This paper continues the studies of vesicle-bound flavins ('anisotropic flavin chemistry'). It is possible to anchor the flavin nucleus in various modes within the lipids/water interface by means of long aliphatic chains and using different saturated lipids, thereby mimicking the specific binding of the coenzyme to the apoprotein in flavoproteins. Based on absorption spectroscopy and EPR spectroscopy studies we explored the rotational mobility and the microenvironment of membrane-bound amphiflavin radicals. N(5)-unsubstituted amphiflavin radicals exhibit a similarly high disproportionation constant as known from isotropic flavin chemistry. However, reasonable stabilization of the radical was achieved by introduction of an alkyl group in position 5 in the reduced state prior to the one-electron oxidation. Adopting the fine structure of the corresponding EPR spectra as assay for the mobility of the semiquinone, we determined rotational relaxation times ranging from 60 ns in the crystalline state down to 10 or 15 ns in the liquid-crystalline state of the membrane. The solvatochromic effect shown by absorption spectra of the membrane-bound flavin radicals reflects a dielectric constant of the microenvironment of c = 30-40, corresponding to the lipid/water interface region. The results obtained in this study are consistent with those obtained previously, from fluorescence analyses, supporting our former conclusions.

Ligand Binding to the blue copper center of horse liver alcohol dehydrogenase

The catalytic zinc ion of horse liver alcohol dehy- drogenase (LADH) is liganded by 2 thiol groups (Cys 46 and 174) and one imidazole nitrogen (His 67) its fourth ligand being water [ 11. Incorporation of Cu*+ into the catalytic zinc binding site of horse liver alcohol dehydrogenase (EC 1 .l .l .l) produces a metastable cupric protein with optical and EPR spectra reminiscent of the so-called ‘blue’ copper proteins [2]. The blue Cu*+ center in cupric alcohol dehydrogenase differs how- ever from the known blue protein metal centers since at least one co-ordination site is occupied by a water molecule. This water molecule is in rapid exchange with solvent water [3]. Small ligand molecules such as pyrazole, an ethanol-competitive inhibitor of the zinc enzyme, are also able to coordinate to the copper ion [2]. This makes possible the use of the copper LADH system as a model not only for the study of structural features of blue cupric centers but also of its chemical behaviour, e.g., the redox reaction mechanisms. Here, we describe results of EPR and optical absorption spectroscopy studies of several complexes of Cu-LADH with small anionic ligand molecules, such as cyanide, azide, imidazole and 2-mercaptoethanol. These com- plexes were found to be metastable and their EPR spectra were indicative of a geometry more tetragonal than in the unliganded cupric enzyme. Binding of NAD+ and NADH to the cupric enzyme gave rise to EPR spectra similar to those observed in ‘blue’ copper centers. 2.

EPR, NMR and absorption spectroscopy studies of Ni(II) and Cu(II) complexes with Thr-Lys-Ala-Ala peptide in aqueous solution

The PMR, EPR and absorption spectroscopy methods have been used to propose the structure of Ni(II) and Cu(II)ThrLysAlaAla complexes formed in the aqueous solution over a broad pH range. The binding sites in tetrapeptide to both metal ions are shown to be the NH 2-Thr, and three deprotonated nitrogens of peptide linkages. In the case of Ni(II) ions the cooperative interaction was observed.

EPR-spectroscopic studies of oxygen adsorption by plasma-activated SiO 2 surfaces

By plasma treatment of SiO 2 surfaces paramagnetic centres are produced showing significant changes in signal intensity after O 2 adsorption. Investigations of O 2 adsorption in its dependence on the degree of coverage, temperature and microwave power implies the formation of new paramagnetic centres at low degrees of O 2 coverage and changes in relaxation times especially at high degrees of oxygen coverage.

Spectroscopic Evaluation and Classification of the Normal, Aging, and Cataractous Lens. (With 1 color plate)

Fluorescence, UV and visible and EPR spectroscopy studies were performed on normal human lenses ranging in age from 1 day to 92 years and on various cataractous lenses. These experiments revealed two specific age-related fluorescent compounds which develop in the lens nucleus on the basis of a UV-induced free radical tryptophan photo-degradation reaction resulting in an increasing concentration of the fluorogen with age. These fluorogens are responsible for the yellow color of the normal lens nucleus which becomes readily apparent by the second decade of life. The increase in the intensity of the yellow color with age parallels the increase in the 360 nm fluorogen concentration; the 435 nm fluorogen which appears to be a secondary product of the former, is related to the hazel to brown color which becomes manifest in the old lenses. The two fluorogens are markedly elevated in the nuclear (brown) cataracts but remain at normal or below normal levels in the cortical cataracts. A classification of the normal lens and primary cataracts is proposed on the basis of UV and visible light transmission and fluorescence spectroscopy in such lenses.