Electron paramagnetic resonance studies on oxidative polymerization mechanism of furan derivatives

The oxidative polymerization of furan, 3-methylfuran and 2,2′,5′,2″-terfuran, induced by iodine in organic solvents having different donicity, was investigated by time-resolved electron paramagnetic resonance (EPR) spectroscopy. The reaction was monitored starting from the onset of the process and radical species arising from the monomers in early stages of the polymerization reaction were detected. The comparison between experimental and theoretical hyperfine coupling constants, obtained by simulated EPR spectra and theoretical ab initio calculations, respectively, allowed one to structurally characterize the above radical species, thus enabling mechanistic conclusions about oxidative coupling. Moreover, the analysis of the time evolution of EPR spectrum during the oxidative polymerization process was used for evaluating the relative amount of localized and mobile radical species along the polymer backbone, thus getting preliminary information about conjugation extent and electroconducting properties of the final material. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36: 1901–1910, 1998     

Modeling the active site of cytochrome oxidase: synthesis and characterization of a cross-linked histidine-phenol

A cross-linked histidine-phenol compound was synthesized as a chemical analogue of the active site of cytochrome c oxidase. The structure of the cross-linked compound (compound 1) was verified by IR, (1)H and (13)C NMR, mass spectrometry, and single-crystal X-ray analysis. Spectrophotometric titrations indicated that the pK(a) of the phenolic proton on compound 1 (8.34) was lower than the pK(a) of tyrosine (10.1) or of p-cresol (10.2). This decrease in pK(a) is consistent with the hypothesis that a cross-linked histidine-tyrosine may facilitate proton delivery to the binuclear site in cytochrome c oxidase. Time-resolved optical absorption spectra of compound 1 at room temperature, generated by excitation at 266 nm in the presence and absence of dioxygen, indicated a species with absorption maxima at approximately 330 and approximately 500 nm, which we assign to the phenoxyl radical of compound 1. The electron paramagnetic resonance (EPR) spectra of compound 1, obtained after UV photolysis, confirmed the generation of a paramagnetic species at low temperature. Because the cross-linked compound lacks beta-methylene protons, the EPR line shape was dramatically altered when compared to that of the tyrosyl radical. However, simulation of the EPR line shape and measurement of the isotropic g value was consistent with a small coupling to the imidazole nitrogen and with little spin density perturbation in the phenoxyl ring. The ground-state Fourier transform infrared (FT-IR) spectrum of compound 1 showed that addition of the imidazole ring perturbs the frequency of the tyrosine ring stretching vibrations. The difference FT-IR spectrum, associated with the oxidation of the cross-linked compound, detected significant perturbations of the phenoxyl radical vibrational bands. We postulate that phenol oxidation produces a small delocalization of spin density onto the imidazole nitrogen of compound 1, which may explain its unique optical spectral properties.     

Influence of conformation on the EPR spectrum of 5,5-dimethyl-1-hydroperoxy-1-pyrrolidinyloxyl: a spin trapped adduct of superoxide

Spin trapping, a technique used to characterize short-lived free radicals, consists of using a nitrone or nitroso compound to "trap" an unstable free radical as a long-lived aminoxyl that can be characterized by EPR spectroscopy. The resultant aminoxyl exhibits hyperfine splitting constants that are dependent on the spin trap and the free radical. Such is the case with 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO-OH) and 2,2-dimethyl-5-hydroperoxy-1-pyrrodinyloxyl (DMPO-OOH) whose hyperfine splitting constants, A(N) = A(H) = 14.9 G and A(N) = 14.3 G, A(H)(beta) = 11.7 G, and A(H)(gamma) = 1.25 G, respectively, have been used to demonstrate the generation of HO(*) and O(2)(*)(-). However, to date, the source of the apparent A(H)(gamma) hyperfine splitting in DMPO-OOH is not known. We consider three possible explanations to account for the unique EPR spectrum of DMPO-OOH. The first is that the gamma-splitting arises from one of the hydrogen atoms at either carbon 3 or carbon 4 of DMPO-OOH. The second is that the gamma-splitting originates from the hydrogen atom of DMPO-OOH. The third is that the conformational properties of DMPO-R change upon going from DMPO-OH to DMPO-OOH. Experimental and theoretical chemical approaches as well as EPR spectral modeling were used to investigate which of these hypotheses may explain the asymmetric EPR spectrum of DMPO-OOH. From these studies it is shown that the 12-line EPR spectrum of DMPO-OOH results not from any proximal hydrogen, but from additional conformers of DMPO-OOH. Thus, the 1.25 G hyperfine splitting, which has been assigned as a gamma-splitting, is actually from two individual EPR spectra associated with different conformers of DMPO-OOH.     

The generation of the diisopropyl nitroxide radical during the reaction of lithium diisopropylamide with α-fluoroacetate esters

The formation of an EPR active species was observed when lithium diisopropylamide (LDA) was employed as the base for deprotonation of fluoroacetates. The EPR of a glass prepared from the reaction mixture showed an intense signal. The solution EPR spectrum of the sample was independent of temperature, and exhibited a hyperfine structure which allowed identification of the radical species as diisopropyl nitroxide. Generation of the enolate by metalation of ethyl fluoroidoacetate gave no evidence for autoxidation occurring in the absence of diisopropylamine.     

Conjugated chromophore arrays with unusually large hole polaron delocalization lengths

We report variable temperature X-band EPR spectroscopic data for the cation radical states of meso-to-meso ethyne-bridged (porphinato)zinc(II) (PZnn) oligomers. These [PZn2-PZn7]+ species span an average 18-75 A length scale and display peak-to-peak EPR line widths (DeltaBp-p) that diminish with conjugation length. Analysis of these EPR data show that PZnn+ structures possess the largest hole polaron delocalization lengths yet measured; experiments carried out over a 4-298 K temperature domain demonstrate remarkably that the charge delocalization length remains invariant with temperature. These cation radical EPR data are well described by a stochastic, near barrierless, one-dimensional charge hopping model developed by Norris for N equivalent sites on a polymer chain, where the theoretical EPR line width is given by DeltaBp-p(N-mer) = (1/N1/2)DeltaBp-p(monomer); PZnn+ oligomers are the first such systems to verify a Norris-type hole delocalization mechanism over a substantial ( approximately 75 A) length scale. Given the time scale of the EPR measurement, these data show that either (i) Franck-Condon effects are incapable of driving charge localization in [PZn2-PZn7]+, resulting in cation radical wave functions which are globally delocalized over a spatial domain that is large with respect to established benchmarks for hole-doped conjugated materials, or (ii) polaron hopping rates in these oligomers exceed 107 s-1, even at 4 K. Finally, this study demonstrates that polymeric building blocks having low magnitude inner sphere reorganization energies enable the development of electronic materials having long polaron delocalization lengths.     

Reaction of superoxide radical with quinone molecules

When the superoxide radical O(2)(?-) is generated on reaction of KO(2) with water in dimethyl sulfoxide, the decay of the radical is dramatically accelerated by inclusion of quinones in the reaction mix. For quinones with no or short hydrophobic tails, the radical product is a semiquinone at much lower yield, likely indicating reduction of quinone by superoxide and loss of most of the semiquinone product by disproportionation. In the presence of ubiquinone-10, a different species (I) is generated, which has the EPR spectrum of superoxide radical. However, pulsed EPR shows spin interaction with protons in fully deuterated solvent, indicating close proximity to the ubinquinone-10. We discuss the nature of species I, and possible roles in the physiological reactions through which ubisemiquinone generates superoxide by reduction of O(2) through bypass reactions in electron transfer chains.     

On the nature of radicals formed in methanol catalytic oxidation

The quantum-chemical calculations of the hydroxymethyl radical •CH₂OH were performed for the first time and a theoretical EPR spectrum of this radical was constructed. The formation of the hydroxymethyl radical in the reaction of methanol oxidation is thermodynamically favorable. The shape and parameters of the constructed spectrum differed from those for radicals experimentally detected in the catalytic oxidation of methanol using the matrix isolation method. However, they are consistent with the spectrum ascribed to the EPR spectrum of •CH₂OH observed in the direct photolysis of methanol. This result allows one to refine the identification of the nature of radicals formed in the catalytic reaction of methanol oxidation.     

Decomposition study of the electron paramagnetic resonance spectrum of irradiated alanine

Recent Electron Paramagnetic Resonance (EPR) studies on alanine powders as a function of irradiation dose and temperature on the one hand and single crystal Electron Nuclear DOuble Resonance (ENDOR) studies on the other hand, showed the presence of at least three radicals contributing to the total alanine EPR spectrum. The latter spectrum obtained after irradiation at room temperature (RT), is dominated by the well-known stable-alanine-radical (SAR) CH3C*HCOO-, also denoted R1. Appropriate heating of irradiated alanine causes the relative contribution of R1 to decrease, resulting in a spectrum mainly caused by the H-abstraction radical CH3C*(NH3)COO-, denoted R2. Although the EPR spectrum of these two radicals could be satisfactorily simulated, their influence on dose reconstruction has not been reported yet. Therefore, a detailed Maximum Likelihood Common Factor Analysis (MLCFA) study has been performed on EPR spectra from polycrystalline alanine samples, after irradiation and heat treatments. Conclusions concerning the number of contributing radicals and their influence on the RT irradiated alanine EPR spectrum will be made.     

Electron paramagnetic resonance of ultraviolet-irradiated polyolefins

If films of polyolefins are ultraviolet-irradiated at liquid nitrogen temperature, alkyl radicals which can be examined by EPR are produced. No EPR spectra are observed from polyolefins irradiated at room temperature in air. Ultraviolet irradiation of polyolefins containing small alkyl side chains generally produces radicals corresponding to the pendant group or methyl radicals if the side chains contain a methyl branch. For some polymers the radical species could not be identified with certainty. Stabilization studies indicate that an optimum concentration of ultraviolet stabilizer is necessary for maximum stabilization of polyolefins. Preliminary results of EPR studies of the ultraviolet irradiation of various polyolefins are given, and some possible radical species are discussed.     

Formation of phenoxy and cyclopentadienyl radicals from the gas-phase pyrolysis of phenol

The formation of radicals from the gas-phase pyrolysis of phenol over a temperature range of 400-1000 degrees C was studied using the technique of low temperature matrix isolation electron paramagnetic resonance (LTMI EPR). Cooling the reactor effluent in a CO2 carrier gas to 77 K produces a cryogenic matrix that exhibits complex EPR spectra. However, annealing by slowly raising the matrix temperature yielded well-resolved, identifiable spectra. All annealed spectra over the temperature range of 700-1000 degrees C resulted in the generation of EPR spectra with six lines, hyperfine splitting constant approximately 6.0 G, g = 2.00430, and peak-to-peak width approximately 3 G that was readily assignable, based on comparison with the literature and theoretical calculations, as that of cyclopentadienyl radical. Annihilation procedures along with microwave power saturation experiments helped to clearly identify phenoxy radicals in the same temperature region. Conclusive identifications of cyclopentadienyl and phenoxy radicals were based on pure spectra of these radicals under the same experimental conditions generated from suitable precursors. Cyclopentadienyl is clearly the dominant radical at temperatures above 700 degrees C and is observed at temperatures as low as 400 degrees C. The low-temperature formation is attributed to heterogeneous initiation of phenol decomposition under very low pressure conditions. The high cyclopentadienyl to phenoxy ratio was consistent with the results of reaction kinetic modeling calculations using the CHEMKIN kinetic package and a phenol pyrolysis model adapted from the literature.     

Assignment of the EPR spectrum of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) superoxide spin adduct

[structure: see text] Spin trapping consists of using a nitrone or a nitroso compound to "trap" an unstable free radical as a long-lived nitroxide that can be characterized by electron paramagnetic resonance (EPR) spectroscopy. The formation of DMPO-OOH, the spin adduct resulting from trapping superoxide (O(2)(*)(-)) with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), has been exploited to detect the generation of superoxide in a wide variety of biological and chemical systems. The 12-line EPR spectrum of DMPO-OOH has been either reported or mentioned in more than a thousand papers. It has been interpreted as resulting from the following couplings: A(N) approximately 1.42 mT, A(H)beta approximately 1.134 mT, and A(H)gamma(1H) approximately 0.125 mT. However, the DMPO-OOH EPR spectrum has an asymmetry that cannot be reproduced when the spectrum is calculated considering a single species. Recently, it was proposed that the 0.125 mT splitting was misassigned and actually results from the superimposition of two individual EPR spectra associated with different conformers of DMPO-OOH. We have prepared 5,5-dimethyl-[3,3-(2)H(2)]-1-pyrroline N-oxide (DMPO-d(2)), and we showed that the EPR spectrum of the corresponding superoxide spin adduct is composed of only six lines, in agreement with the assignment of the 0.125 mT splitting to a gamma-splitting from a hydrogen atom bonded to carbon 3 of DMPO. This result was supported by DFT calculations including water solvation, and the asymmetry of the DMPO-OOH EPR spectrum was nicely reproduced assuming a chemical exchange between two conformers.     

Comparative identification of irradiated herbs by the methods of electron paramagnetic resonance and thermoluminescence

Non irradiated and γ-irradiated dry herbs savoury (Savoury), wild thyme (Thymus serpollorium) and marjoram (Origanum) with absorbed dose of 8 kGy have been investigated by the methods of elecrtron paramagnetic resonance (EPR) and thermoluminescence (TL). Non-irradiated herbs exhibit only one weak siglet EPR signal whereas in irradiated samples its intensity increase and in addition two satelite lines are recorded. This triplet EPR spectrum is attributed to cellulose free radical generated by irradiation. It has been found that upon keeping the samples under the normal stock conditions the life-time of the cellulose free radical in the examined samples is ∼60–80 days. Thus the conclusion has been made that the presence of the EPR signal of cellulose free radical is unambiguous indication that the sample under study has been irradiated but its absence can not be considered as the opposite evidence. In the case when EPR signal was absent the method of TL has been used to give the final decision about the previous radiation treatment of the sample.     

A SURVEY OF EPR INVESTIGATIONS OF BACTERIAL PHOTOSYNTHESIS

Abstract— Investigations in which EPR has been used as a probe of the mechanism of the primary quantum conversion reaction and/or electron transport reactions in bacterial photosynthesis are surveyed. These investigations include studies of whole cell organisms and simpler sub-cellular preparations, chromatophores and bacteriochlorophyll-protein complexes. Electron paramagnetic resonance studies have successfully demonstrated that the primary donor of the photosynthetic, photochemical reaction involves a dimer of bacteriochlorophyll, generally referred to as P870. P870 is photochemically oxidized to a cation radical which exhibits a g= 2.0025 EPR signal. Comparative studies of the time behaviour of this signal in whole cells and in sub-cellular preparations show that electron flow in the whole cells is substantially different than in the cell-free systems. The primary acceptor molecule of the photochemical reaction has not been conclusively identified. When it is photochemically reduced, it exhibits a broad EPR absorbance centered at g= 1.8 observable only at low temperatures. This signal involves an iron atom and a quinone molecule. Two possible identifications of the species responsible for the g= 1.8 signal are an iron-quinone complex and an iron-sulfur protein. The latter identification would require that one primary acceptor function for two primary donors and that the removal of a tightly held quinone alter the integrity of the system so as to inhibit the photochemistry. When the primary acceptor is chemically reduced, a photo-induced, polarized triplet EPR spectrum is detected. Both absorption and emission lines are, observed as if only one substate (m= 0) of the triplet manifold is populated. The zero field parameters of the triplet spectrum suggest that the triplet is formed through a decay of a biradical, not through an optical singlet to triplet transition. Low temperature EPR studies of photosynthetic preparations which had been poised at room temperature by subjecting the preparations to different redox potentials and/or dark adaptation and illumination show the presence of a number of light-influenced, EPR active components. The spectral characteristics of these components are indicative of iron-heme (both low and high-spin forms) and iron-sulfur (both oxidized and reduced) proteins and at least one other organic free radical distinct from P870⁺. The spectra varied for different strains of bacteria. Also, some of the signals detected in the whole cell organism were not detected in cell free preparations and the kinetics of the light influenced signal amplitudes were different in the whole cells than in chromatophores.     

GENERATION OF FREE RADICALS DURING PHOTOSENSITIZATION OF HYPOCRELLIN A AND THEIR EFFECTS ON CARDIAC MEMBRANES

Hypocrellin A (HA), a peryloquinone derivative, has recently been isolated from a fungus Hypocrella bambusae. This lipid soluble pigment, in combination with phototherapy, has been used to treat many skin diseases including the keloids caused by scalding and burns. We have studied the effects of photosensitized HA on biomembranes using pig heart microsomes. Photosensitization of HA was found to peroxidize the membrane lipids in the cardiac microsomes. The photodamage imposed by HA depended not only on the concentration of HA but also on the time of irradiation and pH of the system. Superoxide dismutase (SOD), ascorbic acid, beta-carotene and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) inhibited the lipid peroxidation approximately 50, approximately 50, approximately 30 and approximately 97%, respectively. Spin trapping in combination with EPR spectroscopic techniques was used to identify the reactive free radicals during the photoreaction. Formation of superoxide anion radical, (O2-.), was identified by the SOD-inhibitable DMPO-O2- EPR spectrum. Both SOD and ascorbic acid inhibited the EPR signal intensity in a dose-dependent manner with rate constants of 6.78 x 10(8) M-1 s-1 and 1.82 x 10(4) M-1 s-1, respectively. The lifetime of O2-., under these conditions, was found to be 1.1 s. Photoirradiation of HA yielded a HA free radical with a g = 2.002 which was not suppressed by SOD but in the presence of reductants such as ascorbic acid and catechol the septum was completely suppressed. The increase of the EPR signal intensity and malondialdehyde formation with increasing pH may be due, in part, to the production of predominant *HA- species at high pH which would be more reactive with oxygen to yield O2-.. These results indicate that the lipid peroxidation of the cardiac membranes observed during photooxidation of HA may arise, in part, from the interaction of membrane lipids with reactive species of oxygen and HA free radical produced during the photo-irradiation.     

An EPR, ENDOR and EIE study of gamma-irradiated poly (lactide-co-glycolide) polymers

Gamma radiation of poly (lactide-co-glycolide) raw polymers and processed microspheres under vacuum and at 77 K results in the formation of a series of free radicals. The resulting powder electron paramagnetic resonance (EPR) spectrum contains a distribution of several different radicals, depending on the annealing temperature, and is therefore difficult to interpret. By utilising the selectivity of the electron nuclear DOuble resonance (ENDOR) and associated ENDOR induced EPR (EIE) techniques, a more direct approach for the deconvolution of the EPR spectrum can be achieved. Using this approach, the radiolytically induced CH3 *CHC(O)R- chain scission radical was identified at 120 K by simulation of the EIE spectrum. At elevated temperatures (250 K), this radical decays considerably and the more stable radicals -O*CHC(O)-, CH3 *C(OR)C(O)- and CH3 *C(OH)C(O)- predominate. This work demonstrates the utility of the EIE approach to supplement and aid the interpretation of powder EPR spectra of radicals in a polymer matrix.     

Two [Fe(IV)=O Trp*] intermediates in M. tuberculosis catalase-peroxidase discriminated by multifrequency (9-285 GHz) EPR spectroscopy: reactivity toward isoniazid

We have characterized the intermediates formed in the peroxidase cycle of the multifunctional heme-containing enzyme KatG of M. tuberculosis. Selected Trp variants from the heme proximal (W321F) and distal (W107F and W91F) sides were analyzed together with the wild-type enzyme with regard to the reaction with peroxyacetic acid and hydrogen peroxide (in the catalase-inactive W107F). The 9 GHz EPR spectrum of the enzyme upon reaction with peroxyacetic acid showed the contribution of three protein-based radical species, two Trp* and a Tyr*, which could be discerned using a combined approach of multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy with selective deuterium labeling of tryptophan and tyrosine residues and site-directed mutagenesis. Trp321, a residue in H-bonding interactions with the iron through Asp381 and the heme axial ligand His270, was identified as one of the radical sites. The 9 GHz EPR signal of the Trp321 radical species was consistent with an exchange-coupled species similar to the oxoferryl-Trp radical intermediate in cytochrome c peroxidase. On the basis of the possibility of distinguishing among the different radical intermediates of the peroxidase cycle in M. tuberculosis KatG (MtKatG), we used EPR spectroscopy to monitor the reactivity of the enzyme and its W321F variant with isoniazid, the front-line drug used in the treatment of tuberculosis. The EPR experiments on the W321F variant preincubated with isoniazid allowed us to detect the short-lived [Fe(IV)=O Por*+] intermediate. Our results showed that neither the [Fe(IV)=O Por*+] nor the [Fe(IV)=O Trp321*+] intermediates were the reactive species with isoniazid. Accordingly, the subsequent intermediate (most probably the other Trp*) is proposed to be the oxidizing species. Our findings demonstrate that the protein-based radicals formed as alternative intermediates to the [Fe(IV)=O Por*+] can play the role of cofactors for substrate oxidation in the peroxidase cyle of KatGs.     

SOME ONE ELECTRON REDUCTION PRODUCTS OF FLAVIN ANALOGS: CYANOISOALLOXAZINES AND DEAZAISOALLOXAZINES

Abstract— 8-Cyanoisoalloxazines have been previously shown to form highly stable radical species at basic pH. We have measured the electron spin resonance (EPR) spectra of the radical forms of 8-cyano-10-methyl-3-sulfopropylisoalloxazine (I) at both acidic and basic pH. In both cases. the EPR spectra are similar to those obtained from unsubstituted isoalloxazines. with no indication of hyperfine splitting due to the cyano nitrogen. Laser photolysis of I in the presence of EDTA at basic pH generates two radical species. One of these decays rapidly by a first-order process to produce thc stable radical. The rate of this decay depends upon the initial flavin concentration, thus suggesting a reaction of the radical with oxidized isoalloxazine. The rates of reaction of the radical species with added oxidants (O₂, ferricyanide), and the pH-dependence of stable radical formation, indicate that the rapidly-decaying species is the anion radical of I, and that the stable radical is formed by its reaction with oxidized flavin. Laser photolysis of I at acidic pH, as well as of 8-cyano-5-deaza-isoalloxazine at acidic or basic pH, does not generate stable radical species. I-Deazaisoalloxazines do not give radical transients at all upon laser photolysis.     

Hyperfine structure observed in EPR spectra of polyphenylacetylene solutions

The high-temperature (>120C) electron paramagnetic resonance (EPR) spectrum of solutions of polyphenylacetylene have been deconvoluted into the spectra of two separate radicals, a delocalized π radical, whose EPR spectrum consists of a single 15-G wide Gaussian line comprising about 90% of the total signal and a second, more localized π radical exhibiting complex hyperfine structure in its EPR spectrum. Some possible structures for the minor component radical are suggested and their hyperfine splitting constants calculated using molecular orbital theory.     

Multifrequency high-field EPR study of the tryptophanyl and tyrosyl radical intermediates in wild-type and the W191G mutant of cytochrome c peroxidase.

Multifrequency (95, 190, and 285 GHz) high-field electron paramagnetic resonance (EPR) spectroscopy has been used to characterize radical intermediates in wild-type and Trp191Gly mutant cytochrome c peroxidase (CcP). The high-field EPR spectra of the exchange-coupled oxoferryl--trytophanyl radical pair that constitutes the CcP compound I intermediate [(Fe(IV)=O) Trp*(+)] were analyzed using a spin Hamiltonian that incorporated a general anisotropic spin-spin interaction term. Perturbation expressions of this Hamiltonian were derived, and their limitations under high-field conditions are discussed. Using numerical solutions of the completely anisotropic Hamiltonian, its was possible to simulate accurately the experimental data from 9 to 285 GHz using a single set of spin parameters. The results are also consistent with previous 9 GHz single-crystal studies. The inherent superior resolution of high-field EPR spectroscopy permitted the unequivocal detection of a transient tyrosyl radical that was formed 60 s after the addition of 1 equiv of hydrogen peroxide to the wild-type CcP at 0 degrees C and disappeared after 1 h. High-field EPR was also used to characterize the radical intermediate that was generated by hydrogen peroxide addition to the W191G CcP mutant. The g- values of this radical (g(x)= 2.00660, g(y) = 2.00425, and g(z)= 2.00208), as well as the wild-type transient tyrosyl radical, are essentially identical to those obtained from the high-field EPR spectra of the tyrosyl radical generated by gamma-irradiation of crystals of tyrosine hydrochloride (g(x)= 2.00658, g(y) = 2.00404, and g(z) = 2.00208). The low g(x)-value indicated that all three of the tyrosyl radicals were in electropositive environments. The broadening of the g(x) portion of the HF-EPR spectrum further indicated that the electrostatic environment was distributed. On the basis of these observations, possible sites for the tyrosyl radical(s) are discussed.     

Automatic fitting to 'powder' EPR spectra of coupled paramagnetic species employing Feynman's theorem

A previous automatic fitting procedure of EPR spectra has been extended with the purpose to characterise coupled paramagnetic complexes in powders and frozen solutions. The theoretical EPR spectra were obtained by matrix diagonalization of a general spin Hamiltonian. A least-squares fitting procedure using analytical derivatives of the calculated spectrum with respect to the spectroscopic, fine structure, nuclear quadrupole, electron-electron, and hyperfine coupling tensors was used to refine those parameters. The powder spectra of matrix isolated *CF3 and RCF2CF2* radicals, previously measured at low temperature, were reanalysed with this method. A theoretically modeled complex consisting of a Cu2+ ion, featuring an axially symmetric g-tensor and 63Cu hyperfine structure anisotropy, and a free radical located at different orientations, with respect to the symmetry axis of the Cu2+ ion, was examined in order to investigate the possibility to recover the magnetic parameters of the separate units and the magnetic couplings between them.