Multifrequency Electron Paramagnetic Resonance Research Articles

Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450

From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-pi-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and Mössbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-pi-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-pi-cation radical, which makes it extremely difficult to trap compound I.

Attempts at Correlation of the Radiolytic Species of Irradiated Solid-State Captopril Studied by Multi-frequency EPR and HPLC

The purpose of this study was to provide insight into the processes that occur after the irradiation of solid-state drugs. Electron paramagnetic resonance (EPR) experiments were performed at two different frequencies, X-band (about 9.5 GHz) and Q-band (about 34 GHz), to identify the radicals present in irradiated captopril. The results confirmed that an irradiated drug can trap several main radicals. Moreover, the radical composition varied as a function of the treatment. In addition, non-volatile final products were studied by liquid chromatography coupled to UV and to mass spectrometry (LC-MS). The variation of the radical composition did not influence the profile of the final products; this appears to indicate that, in the case of captopril, the trapped radicals observed by EPR are not the main precursors of the final products. Finally, high-performance liquid chromatography data appear to indicate that radiosterilization of captopril is feasible.

A Multifrequency Electron Spin Resonance Study of T4 Lysozyme Dynamics Using the Slowly Relaxing Local Structure Model

Electron spin resonance (ESR) spectra were obtained at 250 and 9 GHz for nitroxide-labeled mutants of the protein T4 lysozyme in aqueous solution over a range of temperatures from 2 to 37.5 °C. Two mutants labeled at sites 72 and 131 were studied and compared. The mutant sites are solvent exposed and free of tertiary interactions with other side chains, but the former is at the center of a 5 turn helix, whereas the latter site is on a small two and a half turn helix. The 250 GHz ESR spectra, because of their “fast time scale”, are rather insensitive to the slow overall tumbling motion of the protein. Thus, they are qualitatively different for the two mutants, implying that there are different local dynamics at the two sites. The 9 GHz spectra, which are significantly affected by the overall tumbling and are less sensitive to the internal dynamics, do not show such marked differences between the two sites. The 250 and 9 GHz spectra for each mutant and temperature were simultaneously fit to the slowly relaxing local structure (SRLS) model for slow-motional ESR, using newly developed software. The SRLS model explicitly accounts for the overall tumbling of the protein and the internal modes of motion, which include the motion of the nitroxide side chain (expected to be the same for both mutants) and backbone fluctuations. Very good simultaneous fits are obtained. Whereas two conformers (or spectral components) are typically detected at the lower temperatures, only a single component is observed at the higher temperatures. The significant differences in the high-frequency spectra for the two mutants are readily attributed mainly to a difference in their respective local ordering. That is, site 72 exhibits significantly greater local ordering than does site 131, which is expected from the greater rigidity of the larger helix on which the 72 site is located. The results of this multifrequency study are compared with a previous 9 GHz study. A description of the application of the SRLS model in such a multifrequency study is provided.

Preface: phys. stat. sol. (a) 201/11

We are glad to introduce in this issue of physica status solidi (a) a series of papers which were presented at the international workshop on Defects and Impurities in Crystalline Boron Nitride Compounds, held in Diepenbeek-Hasselt, Belgium, on 20–21 February 2004. Because of the strong scientific connections, this workshop was jointly organised with the 9th International Workshop on Surface and Bulk Defects in CVD Diamond Films (18–20 February 2004) and this led to the joint publication of the contributions in the present issue. For several years boron nitride, and in particular cubic boron nitride, is back in the centre of materials research activities because of new potential applications as a wide band gap semiconductor for electronics and opto-electronics in extreme conditions. However, important efforts are needed to understand the role of defects and impurities in controlling its semiconducting properties and growth processes in order to realise these promises. An international group of researchers (30 participants from 12 countries: Belarus, Belgium, Czech Republic, Germany, Italy, Japan, Latvia, Romania, Russia, Sweden, Ukraine, USA; among which 8 invited) has gathered to discuss the present status of research on the following topics: Impurities and morphology, Synthesis and microstructure, Optical and electrical properties, and Point defects characterisation. Contributions were presented on boron nitride single crystals, on p–n junctions formed with this material, but also on crystalline powders, thin films and B–C–N nanotubes. Among the characterisation methods which have been discussed, we mention optical spectroscopy (absorption, luminescence, Raman), multifrequency electron spin resonance, high resolution and analytical electron microscopy and analytical micro ion beam techniques. This workshop was organised as a final activity of the Flemish–Romanian scientific and technological cooperation project “Defects in diamond-like materials of the B/C/N system”, involving from the Flemish side both the Experimental Condensed Matter Physics Laboratory at the Physics Department of the University of Antwerp and the Materials Physics Division at the Institute for Materials Research of the Limburgs Universitair Centrum, and from the Romanian side the Microstructure of Defects in Solids Laboratory of the National Institute for Materials Physics in Magurele–Bucuresti. We gratefully acknowledge the financial support of the Flemish and Romanian governments through the above mentioned international bilateral project, as well as the organisational support of all three home institutions along the duration of the whole project. We also wish to personally thank Miloš Nesládek and Ken Haenen for the invaluable and continuous assistance in the practical organisation of this workshop.

Multifrequency EPR Study of Metallofullerenes: Eu@C82 and Eu@C74

In this work, the cage structure of a family of europium metallofullerenes, Eu@C74 and three isomers of Eu@C82, were investigated. The analogy of the electronic states of three isomers of Eu@C82 with those of three isomers of Ca@C82 was confirmed by UV−vis−near-IR absorption spectra. The symmetries Cs, C2, and C2v were assigned for three isomers of Eu@C82 by comparing each corresponding isomer of Ca@C82. The D3h cage structure of Eu@C74, which is expected only for the C74 from the isolated pentagon rule, was also confirmed by comparing its photoabsorption spectrum with that of Ca@C74. Multifrequency electron paramagnetic resonance (EPR) spectroscopy was employed to determine the zero-field splitting (ZFS) parameters of the metallofullerenes, which are closely related to the surrounding cage structures. The experimental X- and W-band EPR spectra were completely reproduced by computer simulations based on a spin Hamiltonian considering the ZFS terms up to fourth order. The introduction of the fourth-order ZFS terms into the Hamiltonian resulted in a precise determination of the second-order ZFS terms. A nonvanishing rhombicity parameter E of Eu@C74 demonstrated the reduction of the symmetry from D3h to C2v due to the positioning of Eu2+ ion at the off-center of C74 cage. Moreover, in this work, the semiphenomenological superposition-exchange model (SPEM) was invoked to examine a relationship between the molecular structures and ZFS parameters. All of the experimental ZFS parameters were satisfactory reproduced in terms of the SPEM, leading to confirmation of the molecular symmetries. The semiphenomenological analysis also enabled us to select the most appropriate cage for Eu@C82 with C2 symmetry from among three possible isomers.

Composition-dependent ratio of orbital-to-spin magnetic moment in structurally disorderedFexPt1−xnanoparticles

The ratio of orbital-to-spin magnetic moment ${\ensuremath{\mu}}_{L}^{\mathrm{eff}}/{\ensuremath{\mu}}_{S}^{\mathrm{eff}}$ averaged over the element-specific contributions of Fe and Pt has been measured for 3-nm ${\mathrm{Fe}}_{x}{\mathrm{Pt}}_{1\ensuremath{-}x}$ nanoparticles at room temperature using the multifrequency electron paramagnetic resonance method for different concentrations of Fe. From a detailed $g$-factor analysis we determine that the ratio decreases from ${\ensuremath{\mu}}_{L}^{\mathrm{eff}}/{\ensuremath{\mu}}_{S}^{\mathrm{eff}}=0.049$ for $x=0.43$ to ${\ensuremath{\mu}}_{L}^{\mathrm{eff}}/{\ensuremath{\mu}}_{S}^{\mathrm{eff}}=0.016$ for $x=0.70$ which is much smaller than the bulk iron value $({\ensuremath{\mu}}_{L}^{\mathrm{Fe}}/{\ensuremath{\mu}}_{S}^{\mathrm{Fe}}=0.045).$ The observed concentration dependence is much stronger than the one calculated for ${\mathrm{Fe}}_{x}{\mathrm{Pt}}_{1\ensuremath{-}x}$ bulk samples and reveals likely changes of the confined electronic structure of the nanoparticle system. The ratio ${\ensuremath{\mu}}_{L}^{\mathrm{eff}}/{\ensuremath{\mu}}_{S}^{\mathrm{eff}}$ takes the lowest value at the concentration $(x=0.70)$ where the magnetic anisotropy energy vanishes in bulk alloys. For $xg0.72$ a phase transition from a fcc to the Fe bcc structure occurs resulting in the increased bulk ratio again.

High Field and Multi-Frequency EPR in Single Crystals of Sperm Whale Met-Myoglobin: Determination of the Axial Zero-Field Splitting Constant and Frequency Dependence of the Linewidth

Electron paramagnetic resonance (EPR) measurements for frequencies from 28.5 GHz to 608.1 GHz and magnetic field up to 14 T have been performed on single crystals of sperm whale high-spin ( S =5/2) met-myoglobin. The EPR resonance field along the c * -axis deviates from the g =5.71 straight line at high frequencies. The axial zero-field splitting constant ( D ) of the met-myoglobin sample is evaluated to be 9.47±0.05 cm -1 by analyzing the resonance fields with the S =5/2 spin Hamiltonian including the D term. The angular dependence of EPR spectra in the a b plane has been also investigated at high frequencies. Two kinds of EPR spectra are observed corresponding to two kinds of different heme sites in the unit cell. A notable change in the linewidth of the spectrum along the c *-axis occurs above 350 GHz, suggesting that the dominant relaxation process changes around 350 GHz. The origins of the linewidth are discussed.

The Use of Multi-frequency EPR Techniques to Identify the Radicals Produced in Irradiated β-Blockers

The identification of radicals trapped in irradiated drugs can be very intricate. A multi-frequency electron paramagnetic resonance (EPR) study is proposed to resolve this problem. The Q-band (ca. 34 GHz) comparison with X-band (ca. 9 GHz) did not show significant differences for the four β-blockers studied (atenolol, esmolol, nadolol and propranolol). The use of a higher frequency (285 GHz) was required. It enabled us to determine the g-tensor values of the radicals present in atenolol and esmolol, respectively, g1=2.0086, g2=2.0059 and g3=2.0021 and g1=2.0066, g2=2.0044 and g3=2.0021. The latter was assigned as a phenoxyl radical, which can not be the case for the former. Therefore, radicals produced in esmolol may result from a more complex mechanism than the abstraction followed by the diffusion of an H atom inside the solid. In addition, two molecules as similar as atenolol and esmolol hydrochloride do not contain the same radicals after irradiation. These two conclusions drawn from the EPR results on β-blockers show clearly the importance of continuing the investigations on radiolytic mechanisms in solid-state drugs.

Antiferromagnetic Resonance Studies on the Spin-Canted Molecular Magnet β-p-NCC6F4CNSSN

Multifrequency (24−94 GHz) and variable temperature (5−300 K) single-crystal electron paramagnetic resonance (EPR) studies have been used to probe the magnetic exchange pathway, spin dynamics, and ...

Characterization of a flavin radical product in a C57M mutant of a LOV1 domain by electron paramagnetic resonance.

In the flavin mononucleotide-binding LOV1 domain of the Phot1-receptor from Chlamydomonas reinhardtii the photoreactive cysteine C57 has been replaced by methionine. Photoexcitation of this C57M mutant yields a metastable photoproduct (C57M-415) that thermally decomposes into a stable paramagnetic species (C57M-675) with extremely red-shifted absorption in the visible range. In this contribution, we describe the characterization of this radical by multi-frequency electron paramagnetic resonance and electron-nuclear double resonance. The main features of the spectra identify the paramagnetic species as a flavin neutral radical. However, detailed analysis shows that the isoalloxazine moiety of the flavin is alkyl substituted at N(5), rather than protonated as is usually the case. The implication of these observations on the likely mechanism of photoproduct generation in wild-type LOV domains is discussed.

Multifrequency electron spin resonance detection of solid-state organic free radicals in HCN polymer and a Titan tholin.

Macromolecules derived from hydrogen cyanide (HCN) may be major components of the dark matter observed in bodies in the outer Solar System, which include comets and asteroids. HCN oligomers and polymers are readily formed at room temperature and react with water to produce polypeptides and alpha-amino acids or undergo pyrolysis to produce nitrogen heterocycles. Electron spin resonance (ESR) spectroscopy shows that HCN polymer mixtures contain a significant amount of long-lived organic free radicals that are primarily carbon-based. For comparison, we have also examined samples of tholins produced from experimental analogs of Titan aerosols, which has been shown by trace organic analysis to consist partly of HCN polymer. The "Titan tholin" exhibits at least two ESR signals that can be assigned to nitrogen- and carbon-centered radicals, although heating the sample eliminates the nitrogen centers and increases the signal from the carbon centers. This result suggests that the nitrogen-centered radicals may be thermodynamically less stable, but are kinetically trapped during the spark-discharge reactions that produce tholins from mixtures of gases such as methane and nitrogen. The results strongly support previous proposals of free radical mechanisms for HCN polymerization.

Probing Local Dynamics of the Photosynthetic Bacterial Reaction Center with a Cysteine Specific Spin Label

A multifrequency electron paramagnetic resonance (EPR) approach was used to probe the dynamic structure of the bacterial photosynthetic reaction center (RC) protein. We have demonstrated that the cysteine specific nitroxide spin label MTSL can be covalently bound to a surface cysteine residue of Rhodobacter sphaeroides RC protein. We suggest that the MTSL nitroxide is bound to an accessible cysteine residue, H156, which is located on the surface of the protein on the H-subunit. Analysis of the multifrequency EPR spectra of the spin-labeled RC proteins suggests the restricted character of the protein dynamics. These dynamics can be described as fast libration in a cone with a correlation time faster than 10-9 s. Several dynamically nonequivalent sites were observed in the EPR spectra, which may reflect distinct conformational substates of local protein structure. This work provides a foundation for future studies with the goal of correlating protein motions with photosynthetic charge-transfer reactions.

Asymmetric binding of the high-affinity Q(H)(*)(-) ubisemiquinone in quinol oxidase (bo3) from Escherichia coli studied by multifrequency electron paramagnetic resonance spectroscopy.

Ubiquinone-2 (UQ-2) selectively labeled with (13)C (I =(1)/(2)) at either the position 1- or the 4-carbonyl carbon is incorporated into the ubiquinol oxidase bo(3) from Escherichia coli in which the native quinone (UQ-8) has been previously removed. The resulting stabilized anion radical in the high-affinity quinone-binding site (Q(H)(*)(-)) is investigated using multifrequency (9, 34, and 94 GHz) electron paramagnetic resonance (EPR) spectroscopy. The corresponding spectra reveal dramatic differences in (13)C hyperfine couplings indicating a strongly asymmetric spin density distribution over the quinone headgroup. By comparison with previous results on labeled ubisemiquinones in proteins as well as in organic solvents, it is concluded that Q(H)(*)(-) is most probably bound to the protein via a one-sided hydrogen bond or a strongly asymmetric hydrogen-bonding network. This observation is discussed with regard to the function of Q(H) in the enzyme and contrasted with the information available on other protein-bound semiquinone radicals.

Variable-frequency EPR study of Mn 2+-doped NH 4Cl 0.9I 0.1 single crystal at 9.6, 36, and 249.9 GHz: structural phase transition

Multifrequency electron paramagnetic resonance studies on the Mn 2+ impurity ion in a mixed single crystal NH 4Cl 0.9I 0.1 were carried out at 9.62 (X-band) in the range 120–295 K, at 35.87 (Q-band) at 77 and 295 K, and at 249.9 GHz (far-infrared band) at 253 K. The high-field EPR spectra at 249.9 GHz are well into the high-field limit leading to a considerable simplification of the spectra and their interpretation. Three magnetically inequivalent, but physically equivalent, Mn 2+ ions with their respective magnetic Z-axes oriented along the crystallographic [1 0 0], [0 1 0], [0 0 1] axes were observed. Simultaneous fitting of EPR line positions observed at X-, Q-, and far infra-red bands was performed using a least-squares procedure and matrix diagonalization to estimate accurately the Mn 2+ spin-Hamiltonian parameters. The temperature variation of the linewidth and peak-to-peak intensities of the EPR lines indicate the presence of λ-transitions in the mixed NH 4Cl 0.9I 0.1 crystal at 242 and 228 K consistent with those observed in the pure NH 4Cl and NH 4I crystals, respectively. A superposition-model analysis of the spin-Hamiltonian parameters reveals that the local environment of the Mn 2+ ion is considerably reorganized to produce axially symmetric crystal fields about the respective Z-axes of the three magnetically inequivalent ions as a consequence of the vacancy created due to charge-compensation when the divalent Mn 2+ ion substitutes for a monovalent NH 4 + ion in the NH 4Cl 0.9I 0.1 crystal. This reorganization is almost the same as that observed in NH 4Cl and NH 4I single crystals, although the latter two are characterized by different, simple cubic and face-centered cubic, structures.

Characterization of Fe−MCM-41 Molecular Sieves with Incorporated Carotenoids by Multifrequency Electron Paramagnetic Resonance

Multifrequency electron paramagnetic resonance (EPR) spectroscopy was applied to study Fe(III)−MCM-41 mesoporous molecular sieves with incorporated carotenoids. It was demonstrated that high-frequency/high-field EPR is a promising technique to increase spectral resolution for proper assignment of different Fe3+ sites, which cannot be resolved by the X-band experiments. The broad unresolved at 9 GHz EPR line in the g = 2 region is due to overlapping signals from Fe3+ sites with different zero field parameters. The peak with g = 2.45 is tentatively assigned to aggregated Fe3+. The signal with g = 2.07 can be attributed to Fe3+ coordinated to oxygen atoms on the surface of the pore. A narrow line with gx = gy = 2.003, gz = 1.99, and E/D = 0.3 was attributed to a single Fe3+ site. The X-band and 94 GHz EPR measurements indicated that extraframework iron species at the surface of the mesopores are mostly responsible for carotenoid oxidation in molecular sieves. ENDOR measurements revealed the orientation of 7‘-apo-7‘,7‘-dicyano-β-carotene and canthaxanthin within Fe−MCM-41.

Role of the Coordinating Histidine in Altering the Mixed Valency of Cu A: An Electron Nuclear Double Resonance-Electron Paramagnetic Resonance Investigation

The binuclear Cu A site engineered into Pseudomonas aeruginosa azurin has provided a Cu A-azurin with a well-defined crystal structure and a CuSSCu core having two equatorial histidine ligands, His120 and His46. The mutations His120Asn and His120Gly were made at the equatorial His120 ligand to understand the histidine-related modulation to Cu A, notably to the valence delocalization over the CuSSCu core. For these His120 mutants Q-band electron nuclear double resonance (ENDOR) and multifrequency electron paramagnetic resonance (EPR) (X, C, and S-band), all carried out under comparable cryogenic conditions, have provided markedly different electronic measures of the mutation-induced change. Q-band ENDOR of cysteine C β protons, of weakly dipolar-coupled protons, and of the remaining His46 nitrogen ligand provided hyperfine couplings that were like those of other binuclear mixed-valence Cu A systems and were essentially unperturbed by the mutation at His120. The ENDOR findings imply that the Cu A core electronic structure remains unchanged by the His120 mutation. On the other hand, multifrequency EPR indicated that the H120N and H120G mutations had changed the EPR hyperfine signature from a 7-line to a 4-line pattern, consistent with trapped-valence, Type 1 mononuclear copper. The multifrequency EPR data imply that the electron spin had become localized on one copper by the His120 mutation. To reconcile the EPR and ENDOR findings for the His120 mutants requires that either: if valence localization to one copper has occurred, the spin density on the cysteine sulfurs and the remaining histidine (His46) must remain as it was for a delocalized binuclear Cu A center, or if valence delocalization persists, the hyperfine coupling for one copper must markedly diminish while the overall spin distribution on the CuSSCu core is preserved.

Some recent multi-frequency electron paramagnetic resonance results on systems relevant for dosimetry and dating

Electron Paramagnetic Resonance (EPR) applications like e.g. EPR dosimetry and dating, are usually performed at X-band frequencies because of practical reasons (cost, sample size, etc.). However, it is increasingly recognized that the radiation-induced EPR signals are strongly composite, what might affect dose/age estimates. A few recent examples from both the dosimetry and dating field, illustrating the problems, will be presented. The involved spectra are mainly due to carbonate-derived radicals ( CO 2 −, CO 3 3−, etc.). Measurements at higher microwave frequencies are often recommended to improve the insight into the spectra and/or the practical signal quantification. Recent results at Q- and W-band frequencies will show that a multi-frequency approach indeed opens many interesting perspectives in this field but also that each frequency may have specific (dis)advantages depending on the EPR probe and application involved. The discussion will concern carbonate-containing apatite single crystals, shells, modern and fossil tooth enamel.

Structural investigation of oxidized chlorosomes from green bacteria using multifrequency electron paramagnetic resonance up to 330 GHz.

Chemical oxidation of the chlorosomes from Chloroflexus aurantiacus and Chlorobium tepidum green bacteria produces bacteriochlorophyll radicals, which are characterized by an anomalously narrow EPR signal compared to in vitro monomeric BChl c (.+) [Van Noort PI, Zhu Y, LoBrutto R and Blankenship RE (1997) Biophys J 72: 316-325]. We have performed oxidant concentration and temperature-dependent X-band EPR measurements in order to elucidate the line narrowing mechanism. The linewidth decreases as the oxidant concentration is increased only for Chloroflexus indicating that for this system Heisenberg spin exchange is at least partially responsible for the EPR spectra narrowing. For both species the linewidth is decreasing on increasing the temperature. This indicates that temperature-activated electron transfer is the main narrowing mechanism for BChl radicals in chlorosomes. The extent of the electron transfer process among different BChl molecules has been evaluated and a comparison between the two species representative of the two green bacteria families has been made. In parallel, high frequency EPR experiments have been performed on the oxidized chlorosomes of Chloroflexus and Chlorobium at 110 and 330 GHz in the full temperature range investigated at X-band. The g-tensor components obtained from the simulation of the 330 GHz EPR spectrum from Chlorobium show the same anisotropy as those of monomeric Chl a (.+) [Bratt PJ, Poluektov OG, Thurnauer MC, Krzystek J, Brunel LC, Schrier J, Hsiao YW, Zerner M and Angerhofer A (2000) J Phys Chem B 104: 6973-6977]. The spectrum of Chloroflexus has a nearly axial g-tensor with reduced anisotropy compared to Chlorobium and monomeric Chl a in vitro. g-tensor values and temperature dependence of the linewidth have been discussed in terms of the differences in the local structure of the chlorosomes of the two families.

Multi-frequency EPR study of radiation-induced radicals in tooth enamel

Human tooth enamel powders, unheated as well as heated prior to X -irradiation at room temperature, have been investigated by means of Q - and W -band Electron Paramagnetic Resonance (EPR). Upon irradiation of enamel, carbonate-derived radicals are generated. The simplest acquired EPR spectra in this study consist mainly of a group of three different \\hbox{CO}_3^{3-} signals, with a very weak \\hbox{CO}_2^{-} contribution. The characterisation of the paramagnetic species in enamel is quite important for the reliability of EPR applications ( e.g. , EPR retrospective dosimetry). The spectra from the heated samples reveal a striking resemblance with spectra from certain irradiated synthetic apatite powders. The spin Hamiltonian parameters obtained from the computer simulations of the Q - and W -band spectra are compared with those reported in the literature.

Estimation of higher-order magnetic spin interactions of Fe(III) and Gd(III) ions doped in α-alumina powder with multifrequency EPR

Fe(III) and Gd(III) ions in α-alumina (A12O3) exhibit spin states ofS = 5/2 andS = 7/2 respectively. The magnitude of the zero-field interaction (ZFI) (D = 0.10−0.15 cm−1) gives rise to an inter Kramers doublet splitting in the same order of magnitude as the X-band electron paramagnetic resonance (EPR) quantum (0.3 cm−1). It is demonstrated that through a careful step-by-step analysis and spectral simulation of EPR spectra taken at D-band (130 GHz), Q-band (35 GHz), and X-band (9 GHz) at room temperature, the (relative) sign and magnitude of the ZFI parameters, b20, b40, and b43, can be reliably estimated.