Changes In EPR Spectra Research Articles

Measuring local pH at interfaces from molecular tumbling: A concept for designing EPR-active pH-sensitive labels and probes.

Molecular probes and indicators are broadly employed for pH measurements in bulk media and at interfaces. The underlying physical principle of pH measurements of most of these probes is based on a change in the electronic structure that, for example, results in a shift of the emission peak of the fluorescence probes, changes in NMR chemical shifts due to the affected electronic shielding, or magnetic parameters of pH-sensitive nitroxides as measured by EPR. Here we explore another concept for measuring local protonation state of molecular tags based on changes in rotational dynamics of electron spin-bearing moieties that are readily detected by conventional continuous wave X-band EPR. Such changes are especially pronounced at biological interfaces, such as lipid bilayer membranes, due to the probe interactions with adjacent charges and polarizable dipoles. The concept was demonstrated by synthesizing a series of pH-sensitive nitroxides and spin-labelled phospholipids. EPR spectra of these newly synthesized nitroxides exhibit relatively small - about 0.5 G - changes in isotropic nitrogen hyperfine coupling constant upon reversible protonation. However, spin-labelled phospholipids incorporated into lipid bilayers demonstrated almost 6-fold change in rotational correlation time upon protonation, readily allowing for pKa determination from large changes in EPR spectra. The demonstrated concept of EPR-based pH measurements leads to a broader range of potential nitroxide structures that can serve as molecular pH sensors at the desired pH range and, thus, facilitates further development of spin-labelling EPR methods to study electrostatic phenomena at chemical and biological interfaces.

High temperature phase transition and multiphase state formation in K3H(SO4)2 superprotonic conductor

EPR spectra of K3H(SO4)2 (KHS) doped with VO2+ or Mn2+ recorded at the temperature higher than about 440K are time-dependent and indicate on anomalously slow kinetics of the paraelastic/ferroelastic phase transition which takes place far below the temperature proposed by Baranov et al. (TII=463K). The formation of a multidomain state upon heating below TII is manifested as irreversible changes in EPR spectra after sample cooling to room temperature. The angular dependence of EPR spectra indicates that K3H(SO4)2 undergoes a ferroelastic phase transition between the point groups 2/m and 3¯m. The EPR spectra of KHS doped with VO2+ or Mn2+ are rapidly changed into the pseudo-powder ones after annealing above TI=480K. This points to a decomposition of the KHS crystal and a multiphase state formation at TI=480K, rather than at superprotonic phase transition, which is typical for the majority of compounds of the M3H(XO4)2 type.

Specific features of molecular mobility of paramagnetic probe in chiral biomimetic solutions

Molecular mobility of a nitroxyl radical (as a paramagnetic probe) in methylcyclohexane (MCH) solutions of chiral biomimetic gelators—trifluoroacetylated amino alcohols (synthetic analogues of biological molecules)—has been studied by monitoring thermally induced changes in EPR spectra. The phase state of the systems has been examined by the low-temperature scanning calorimetry method. There has been found an unusual concentration and temperature dependence of probe species mobility in diluted solutions (10–2–10–3 M) of trifluoroacetylated amino alcohols with a molecular weight of no more than 200. From the temperature-induced changes in EPR spectra of the paramagnetic probe in the temperature range 170–290 K, the rotation activation energies of probe species in biomimetic solutions and in the neat solvent. The minimum radical rotation activation energy (2.1 kcal/mol) is observed in the most concentrated gelator solution, whereas in neat MCH, this energy is 3.7 kcal/mol.

Structure, Dynamics, and Electrostatic Effects on Membrane Binding of Nod Peptides

A Sec14p-nodulin domain phosphatidylinositol transfer protein has been shown to polarize membrane growth of root hairs. To understand the mechanism of regulation, we have synthesized three peptides mimicking the sequence thought to be responsible for the targeted membrane binding containing single Cys substitutions. Binding of the peptides to model membranes was studied by spin-labeling EPR, ITC and DLS as a function of the bilayer lipid composition, with emphasis on the presence of phosphatidylinositol (PI) and brain PI(4,5)P2. For EPR studies, peptides were labeled with thiol-specific spin label (2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSL) and membrane binding was characterized from changes in EPR spectra. Binding data were analyzed using a comprehensive model that includes description of electrostatic interaction by the Gouy-Chapman theory. Analysis of local polarity and accessibility of the EPR label for the bound form of the peptides was used to assess the location of bound peptides within model phospholipid bilayers. Substantial differences in binding behavior of these charged peptides were observed despite the similarity in sequences. Binding of the peptides did not induce liposome leakage or fusion. Unusual temperature dependent reversible effect of the peptides on liposome aggregation was observed and showed strong dependence upon membrane composition, especially the presence of PI(4,5)P2 lipids. Supported by NSF 0843632 to TIS.

Preparation and Magnetic Investigation of Magnetic Nanoparticles Entrapped Hydrogels and Its Possible Use as Radiation Shield

Magnetic nanoparticles were fabricated in hydroxyethyl methacrylate-dextran hydrogel as stabilizer by loading co-precipitation technique (HDFeL) and gamma irradiation (HDFeR). The structure and the surface morphology of the hydrogels were characterized by fourier transform infrared spectroscopy and scanning electron microscopy. The swelling study of different hydrogels in bidistilled water and different pH’s confirmed that the hydrogels are highly influenced by changing the pH. The average particle size measurements were demonstrated by transmission electron microscope and dynamic light scattering and it is found to be in the nano scale. EPR results show that the (HDFeL) samples exhibited large magnetization while the (HDFeR) samples exhibited no magnetization. EPR signal consists of two components: a broad component observed at g = 2.05774 and narrow component observed at g = 2.00219. All product samples irradiated at different irradiation doses (1–600 kGy) demonstrate no change in EPR spectra. It is expected that the Fe3O4 nanoparticles could be used for radiation shielding.

Changes in the Molecular Structure of UHMW Polyethylene under the Influence of Thermal and Radiation Modification, as Well as Time

The paper presents the results of an examination of UHMW polyethylene type GUR1050 using the EPR, FTIR and SEM methods. The relationship between the number of radicals in the polymer and the absorbed dose of ionizing radiation has been shown. The radicals, being highly active macroparticles, disappear in time, so that after about a year their amount is reduced by approximately one order of magnitude. The disappearance of the radicals over time results in a change in EPR spectra, an increase in the degree of oxidation, and cross-linking. The most probable mechanisms are the formation of alkyl and allyl radicals and their gradual transitions, which lead to the absorption of atmospheric oxygen, and the recombination of radicals, which leads to the formation of cross linkages. Thermal treatment of the samples is an effective method of eliminating the radicals. Tribological tests and their analysis by means of SEM observation showed that the irradiated samples proved to have higher resistance to abrasion than the material that was not exposed to radiation.

Probing Dielectric and Hydrogen Bonding Gradients in Biological Membranes

Nitroxide spin-labeling in combination with EPR spectroscopy has found many applications in studying structure and dynamics of proteins and biological membranes. Recently, there has been a substantial interest in utilizing EPR to characterize local effects of polarity and hydrogen bonding in proteins and biological membrane systems. Here we report on employing an arsenal of advanced spin-labeling EPR methods to profile heterogeneous dielectric and hydrogen bonding environment along the α-helical chain of an alanine-rich WALP peptide that is anchored in a lipid bilayer in a transmembrane orientation. A series of WALP cysteine mutants was labeled with a pH-sensitive nitroxide IMSTL (S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester) that is similar in molecular volume to phenylalanine. The protonation state of this nitroxide could be directly observed by EPR allowing us to follow proton gradient across the membrane in the vicinity of the WALP α-helix, and, thus, to reconstruct the gradient in the effective dielectric constant. These experiments were complemented by assessing local polarity from characteristic changes in EPR spectra that were enhanced by the use of perdeuterated and 15N-substituted nitroxides and high field EPR at 130 GHz (D-band). Formation of hydrogen bonds between the nitroxides and membrane-penetrating water molecules was observed directly in HYSCORE X-band experiments. Such measurements allowed us to derive experimental profiles of heterogeneous dielectric and hydrogen bonding environment along a typical transmembrane α-helix. Supported by: NSF-0843632 to TIS and NIH 1R01GM072897 to AIS.

Binding of oxindole-Schiff base copper(II) complexes to DNA and its modulation by the ligand

Previous studies on copper(II) complexes with oxindole-Schiff base ligands have shown their potential antitumor activity towards different cells, inducing apoptosis through a preferential attack to DNA and/or mitochondria. Herein, we better characterize the interactions between some of these copper(II) complexes and DNA. Investigations on its binding ability to DNA were carried out by fluorescence measurements in competitive experiments with ethidium bromide, using plasmidial or calf-thymus DNA. These results indicated an efficient binding process similar to that observed with copper(II)-phenanthroline species, [Cu( o-phen) 2] 2+, with binding constants in the range 3 to 9 × 10 2 M − 1 . DNA cleavage experiments in the presence and absence of distamycin, a recognized binder of DNA, indicated that this binding probably occurs at major or minor groove, leading to double-strand DNA cleavage, and being modulated by the imine ligand. Corroborating these data, discrete changes in EPR spectra of the studied complexes were observed in the presence of DNA, while more remarkable changes were observed in the presence of nucleotides (AMP, GMP, CMP or UMP). Additional evidence for preferential coordination of the copper centers to the bases guanine or cytosine was obtained from titrations of these complexes with each nucleotide, monitored by absorption spectral changes. Therefore, the obtained data point out to their action as groove binders to DNA bases, rather than as intercalators or covalent cross-linkers. Further investigations by SDS PAGE using 32P-ATP or 32P-oligonucleotides attested that no hydrolysis of phosphate linkage in DNA or RNA occurs, in the presence of such complexes, confirming their main oxidative mechanism of action.

Supramolecular dimer formation through hydrogen bond extensions of carboxylate ligands – Path for magnetic exchange

A new complex of unusual composition [Cu(3-O 2Nbz) 2(nia)(H 2O) 2] ( 1) (nia = nicotinamide, 3-O 2Nbz = 3-nitrobenzoate) has been prepared and studied together with two other complexes of composition [Cu(4-O 2Nbz) 2(nia) 2(H 2O) 2] ( 2) and [Cu(4-O 2Nbz) 2(nia) 2]∙(4-O 2NbzH) 2 ( 3) (4-O 2Nbz = 4-nitrobenzoate). The composition of all complexes has been determined by elemental analysis, the complexes have been studied by electronic, infrared and EPR spectroscopy, as well as by magnetization measurements over the temperature range 1.8–300 K, and their structures have been solved. The structure of complex ( 1) consists of molecules, where Cu(II) atom is monodentately coordinated by the pair of 3-nitrobenzoato anions in trans-positions together with water and nicotinamide molecules, forming nearly tetragonal basal plane, and by another water molecule in axial position of tetragonal-pyramidal coordination polyhedron. The neighboring molecule coordination polyhedron basal planes are coplanar and allow formation of supramolecular dimers with strong H-bonds between hydrogen atoms from equatorially coordinated water molecules and uncoordinated carboxylate oxygen atoms thus giving the nearest Cu ⋯ Cu distance of 4.886(2) Å. Magnetization measurements showed that complex ( 1) exhibits maximum of magnetic susceptibility at 6.5 K and a fit to Bleaney-Bowers equation gave singlet–triplet energy gap 2 J = −6.25 cm −1, and zJ′ = −0.03 cm −1. This might be an experimental proof that the carboxylate bridges extended with hydrogen bonds are the pathway of the spin–spin interactions. The temperature dependence of changes in EPR spectra of ( 1) and the spectrum at 4.2 K have confirmed its hydrogen bonded dimeric structure. The calculated Cu ⋯ Cu distance 4.8 Å is in very good agreement with the value obtained from crystal structure. The complexes ( 2) and ( 3) at 300 K exhibit magnetic moment μ eff = 1.98 B.M. and μ eff = 1.84 B.M., respectively. These values practically do not change with lowering the temperature up to 5 K and only small drops to μ eff = 1.87 B.M. (for ( 2)) and μ eff = 1.79 B.M. (for ( 3)) at 1.8 K have been observed. The EPR spectra of complex ( 2) at room temperature as well as at 77 K are of axial type with g ⊥ = 2.062 and g || = 2.285 and exhibit resolved parallel hyperfine splitting with A || = 160 Gauss. The EPR spectra of complex ( 3) at room temperature as well as at 77 K are of axial type with g ⊥ = 2.065 and g || = 2.235 and exhibit unresolved parallel hyperfine splitting. EPR spectra of ( 2) and ( 3) are consistent with the X-ray structure.

The effect of dose on light-sensitivity of radicals in alanine EPR dosimeters

In this work we present a study of light-induced effects on free radicals and their transformations in γ-irradiated pure l-alanine and in commercially available alanine detectors: rods, pellets and films. Samples irradiated to doses from 2 Gy to 4000 kGy were exposed to light from a fluorescent lamp and to ordinary daylight. The observed changes in EPR spectra of the samples were analyzed with regard to their intensity and shape. The shape analysis was based on numerical decomposition of the measured spectra into model spectra reflecting contributions of R1, R2 and R3 radical populations in the samples. The illumination of alanine dosimeters resulted in significant decrease of the central EPR line and was accompanied by distinct variations in the shape of EPR spectra. The rate of light-induced decay in spectra amplitude was found to be dependent on dose of ionizing radiation—the sensitivity to light was decreasing with increase in dose in all detectors in the 2–5 × 10 5 Gy dose range. The exposure of γ-irradiated (to 300 Gy) alanine to normal, diffused daylight resulted in decay of the signal amplitude at rate about 0.5% per week. It was shown that decay in the R1 component was responsible for the observed reduction of the spectra amplitude. The observed increase in R2 contributions in samples exposed to light confirmed a hypothesis of R → R2 radical transformations promoted by visible light. The reported effects indicate a necessity of protection of irradiated dosimeters from their prolonged exposure to light.

The Chemistry of Phospholipid Binding by the Saccharomyces cerevisiae Phosphatidylinositol Transfer Protein Sec14p as Determined by EPR Spectroscopy

The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C5 region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer.

EPR Spectra of Some Salts of Bis[η5-1,2-Dicarbollyl]nickeliate(III)(1-) Anion [NiIII (η 5-1,2-B9C2H11)2

Some salt-like complexes of the cluster anion [NiIII (η5-1,2-B9C2H11 )2]− ([NiCb2]−), containing paramagnetic Ni3+ ion, with cations Cs+, (CH3)4N+, [MnPhen3]2+ (where Phen is 1,10-phenanthroline) are studied by EPR method at 77 K and 300 K. A neutral complex [MnPhen2(NCS2] is also studied for comparison. The synthesis procedure and X-ray diffraction analysis of [MnPhen3][NiCb2]2 complex with paramagnetic ions Mn2+ (3d5) and Ni3+ (3d7) are described. The EPR data of isostructural complexes [MnPhen3][NiCb2]2 and [MnPhen3][CoCb2]2 are reported. No exchange or dipole-dipole interaction was observed between two paramagnetic ions (Mn2+ and Ni3+) simultaneously present in a complex structure. The temperature changes in EPR spectra of solid compounds are caused by rearrangements in the Mn2+ surrounding. In the case of a salt with a compact spherical Cs+ ion, the local perturbation in a second coordination sphere of [NiCb2]− anion leads to redistribution of the electron density and changes in g-factor.

Site-directed electrostatic measurements with a thiol-specific ph-sensitive nitroxide: differentiating local pK and polarity effects by high-field EPR.

This communication describes the use of a methanethiosulfonate derivative of an imidazolidine nitroxide, methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethyl-imidazolidin-4-ylmethyl) ester, IMTSL, for site-directed pKa determination of peptides by electron paramagnetic resonance. This spin label is covalently attached to the thiol group of unique cysteines incorporated into peptide structures. The tertiary amine nitrogen N3 of the label readily participates in proton exchange reactions, which are monitored through changes in EPR spectra of nitroxide moiety. Using EPR at 95 GHz (W-band) isotropic magnetic parameters of this nitroxide, both Aiso and giso, were calibrated in solvents of different polarity and pH. Two different linear correlations between Aiso and giso for acidic and basic forms of IMTSL were observed, making it possible to differentiate effects of local polarity from N3 protonation on nitroxide EPR spectra. Titration of a synthetic P11 peptide fragment of the laminin B1 chain illustrates the utility of this method.

Changes of EPR Spectra of Wood Impregnated with Copper-Based Preservatives during Exposure to Several Wood-Rotting Fungi

SummaryThe tolerance of various fungi against copper was examined. For this purpose, we impregnated Norway spruce (Picea abies) specimens with two different aqueous solutions: copper(II) octanoate with ethanolamine or copper(II) sulfate (cCu= 1.0 × 10−2mol/l). Impregnated and unimpregnated test specimens were then exposed to brown rot fungiAntrodia vaillantiiandGloeophyllum trabeumor to white-rot fungiSchizophyllum communeandTrametes versicolor. After 2, 4, 6 and 12 weeks of exposure Electron Paramagnetic Resonance, Atomic Absorption Spectroscopy and mass loss measurements were performed. The results indicate thatA. vaillantii, G. trabeumandT. versicolortransform copper(II) sulfate in wood into non-soluble, and therefore non-toxic, copper oxalate. The intensity of this reaction depends on the amount of excreted oxalic acid and was the highest forA. vaillantiiand the lowest forT. versicolor. In the presence of ethanolamine, formation of insoluble copper oxalate was not possible and therefore, decay could not proceed. The major portion of copper remained in the wood and only minor amounts were in some cases translocated into nutrient media.

Temperature Induced Change in Epr Spectrum of Fe 3+ Ion in the Complex of Iron with 2-Hydroxy-1-Nitrozonaphthalene

In temperature interval T=4.2÷290 K in polycrystalline substance-2-hydroxy-1-nitrozonaphathalene Na[Fe(C 10 H 6 ONO) 3 ], the EPR spectrum of Fe 3+ has been investigated. The EPR spectrum is a superposition of two resonance lines and the non-resonance absorption. At the temperature change the redistribution of the absorption between the resonance lines takes place. The resonance line 1 with the effective g-factor g 1 =4.32±0.02 has a maximal intensity at T=4.2 K. The resonance line 2 with the effective g-factor g 2 =2.00±0.01 has the maximal intensity at T=290 K. With rising of temperature the integral intensity of the line 1 is decreasing, while intensity of the line 2 increasing. The second peculiarity of the temperature changes in the EPR spectrum of Fe 3+ in the Na[Fe(C 10 H 6 ONO) 3 ] is the exponential increasing of the linewidth for resonance line 2 when the temperature is lowered. The properties of EPR spectrum manifested in complex of iron with 2-hydroxy-1-nitrozonaphathalene are characteristics of the magnetic centers with multiple-minimum potential.

An unusual phase transition in the crystal structure and physical properties of (TTF)9[Mo(CN)8]2·4H2O, where TTF = tetrathiafulvalene

At 270 K, the charge transfer salt (TTF)9[Mo(CN)8]2·4H2O, I, crystallizes in the triclinic space group P with a = 9.9094(2), b = 10.6781(2), c = 23.6086(7) A, α = 75.7910(8), β = 88.6010(9), γ = 78.5250(8)°, V = 2372.5(1) A3 and Z = 2. At 120 K, the space group is unchanged with a = 9.7990(7), b = 10.6630(5), c = 22.9940(2) A, α = 79.981(4), β = 89.798(4), γ = 79.013(4)°, V = 2321.5 A3 and Z = 2. On comparing the two sets of data, we see significant changes in the cell parameters, most notably in the angle α. Variable temperature crystallographic studies indicate a first order phase transition accompanied by hysteresis, which corresponds to a change in the transport properties. I is a semiconductor and the high temperature activation energy of 0.06 eV changes sharply to 0.15 eV below 236 K. Bulk magnetic susceptibility and ESR measurements indicate that the TTF molecules are antiferromagnetically coupled. The temperature dependence of the EPR spectrum changes from 300–200 K, in approximate agreement with the transport and structural results. The optical spectrum of (TTF)9[Mo(CN)8]2·4H2O consists of several broad bands assigned to TTF charged molecules, to [Mo(CN)8]4− and to charge transfer from the donors to the acceptor in the near infra-red range. Preliminary magnetic susceptibility measurements under light irradiation with a multi-line (752.5–799.3 nm) laser were also performed, but no photomagnetic effect was noted.

Site-directed Mutagenesis of 2,4-Dichlorophenoxyacetic Acid/α-Ketoglutarate Dioxygenase: IDENTIFICATION OF RESIDUES INVOLVED IN METALLOCENTER FORMATION AND SUBSTRATE BINDING

2,4-dichlorophenoxyacetic acid (2,4-D)/alpha-ketoglutarate (alpha-KG) dioxygenase (TfdA) is an Fe(II)-dependent enzyme that catalyzes the first step in degradation of the herbicide 2,4-D. The active site structures of a small number of enzymes within the alpha-KG-dependent dioxygenase superfamily have been characterized and shown to have a similar HXDX(50-70)HX(10)RXS arrangement of residues that make up the binding sites for Fe(II) and alpha-KG. TfdA does not have obvious homology to the dioxygenases containing the above motif but is related in sequence to eight other enzymes in the superfamily that form a distinct consensus sequence (HX(D/E)X(138-207) HX(10)R/K). Variants of TfdA were created to examine the roles of putative metal-binding residues and the functions of the other seven histidines in this protein. The H167A, H200A, H213A, H245A, and H262A forms of TfdA formed inclusion bodies when overproduced in Escherichia coli DH5alpha; however, these proteins were soluble when fused to the maltose-binding protein (MBP). MBP-TfdA exhibited kinetic parameters similar to the native enzyme. The H8A and H235A variants were catalytically similar to wild-type TfdA. MBP-H213A and H216A TfdA have elevated K(m) values for 2,4-D, and the former showed a decreased k(cat), suggesting these residues may affect substrate binding or catalysis. The H113A, D115A, MBP-H167A, MBP-H200A, MBP-H245A and MBP-H262A variants of TfdA were inactive. Gel filtration analysis revealed that the latter two proteins were highly aggregated. The remaining four inactive variants were examined in their Cu(II)-substituted forms by EPR and electron spin-echo envelope modulation (ESEEM) spectroscopic methods. Changes in EPR spectra upon addition of substrates indicated that copper was present at the active site in the H113A and D115A variants. ESEEM analysis revealed that two histidines are bound equatorially to the copper in the D115A and MBP-H167A TfdA variants. The experimental data and sequence analysis lead us to conclude that His-113, Asp-115, and His-262 are likely metal ligands in TfdA and that His-213 may aid in catalysis or binding of 2,4-D.

Reversible alkaline inactivation of lignin peroxidase involves the release of both the distal and proximal site calcium ions and bishistidine co-ordination of the haem

Phanerochaete chrysosporium lignin peroxidase isoenzyme H2 (LiP H2) exhibits a transition to a stable, inactive form at pH 9.0 with concomitant spectroscopic changes. The Söret peak intensity decreases some 55% with a red shift from 408 to 412 nm; the bands at 502 nm and 638 nm disappear and the peak at 536 nm increases. The EPR spectrum changes from a signal typical of high spin ferric haem to an exclusively low spin spectrum with g=2.92, 2.27, 1.50. These data indicate that the active pentaco-ordinated haem is converted into a hexaco-ordinated species at alkaline pH. Room temperature near-IR MCD data coupled with the EPR spectrum allow us to assign the haem co-ordination of alkali-inactivated enzyme as bishistidine. Re-acidification of the alkali-inactivated enzyme to pH 6 induces further spectroscopic changes and generates an irreversibly inactivated species. By contrast, a pH shift from 9.0 to 6.0 with simultaneous addition of 50 mM CaCl(2) results in the recovery of the initial activity together with the spectroscopic characteristics of the native ferric enzyme. Incubating with 50 mM CaCl(2) at a pH between 6.0 and 9.0 can also re-activate the enzyme. Divalent metals other than Ca(2+) do not result in restoration of activity. Experiments with (45)Ca indicate that two tightly bound calcium ions per enzyme monomer are lost during inactivation and reincorporated during subsequent re-activation, consistent with the presence of two structural Ca(2+) ions in LiP H2. It is concluded that both the structural Ca(2+) ions play key roles in the reversible alkaline inactivation of LiP H2.

Structure and dynamics of radicalized hydrocarbon derivatives included in perhydrotriphenylene single crystals

The radical obtained by γ-irradiation of the nonadecan-10-one/perhydrotriphenylene (10-NDO/PHTP) inclusion compound has been studied by EPR spectroscopy. The radical is formed by removal of one of the protons in the α position to the carbonyl group and the unpaired electron spin is coupled to the α proton and to two equivalent β protons. The hyperfine (hf) coupling constants vary slowly on lowering the temperature until an abrupt change occurs at T = 150 K. The radical reorients quickly on the EPR timescale inside the PHTP channels. The reorientational correlation time τr is obtained from the linewidth values and it is found to be shorter than the τr value measured previously for the same radical included in urea. The EPR spectrum changes in the timescale of weeks and after two months displays the presence of an additional hf coupling with a pair of equivalent protons, the other spectral parameters being unchanged with respect to those of the freshly irradiated radical. This modification is probably due to a folding of the radicalized guest molecule inside the host channel.

Resonance Raman spectroscopy of nitrile hydratase, a novel iron-sulfur enzyme.

Resonance Raman spectra of Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) nitrile hydratase, a novel non-heme iron enzyme, have a large number of peaks in the 300-500 cm-1 region; observation of shifts in these peaks after labeling with 34S shows that they arise from cysteine coordinated to the ferric ion in the protein. The rich Raman spectra result from coupling of the Fe-S stretch with cysteine side chain deformation modes; the observation of 15N isotope shifts in most of these peaks suggests participation of N-donor metal ligands and peptide backbone amide nitrogens in these modes as well. The aggregate 34S isotope shift is too large to result from a single cysteine ligand, consistent with the analysis of EXAFS data that shows two or three S-donor ligands [Scarrow et al. (1996) Biochemistry 35, 10078-10088]. Widespread 2H isotope shifts seen after exchange of the protein into 2H2O suggest the presence of hydrogen bonds to the coordinated cysteine sulfurs. Comparison of the resonance Raman spectra of nitrile hydratase prepared at pH 7.3 and 9.0 shows a shift of intensity into the higher-energy peaks in the spectra of the latter sample. This is interpreted as resulting from an increase in Fe-S bond strength at the higher pH and is supported by observation of a small decrease in Fe-S bond length in the EXAFS analysis [Scarrow et al. (1996) Biochemistry 35, 10078-10088]. Such a decrease in Fe-S bond length is also consistent with pH dependent changes in EPR spectra and could reflect the loss of one or more hydrogen bonds to sulfur ligands.