Davies Electron-nuclear Double Resonance Research Articles

Polypyridyl-Based Copper Phenanthrene Complexes: Combining Stability with Enhanced DNA Recognition.

We report a series of copper(II) artificial metallo-nucleases (AMNs) and demonstrate their DNA damaging properties and in-vitro cytotoxicity against human-derived pancreatic cancer cells. The compounds combine a tris-chelating polypyridyl ligand, di-(2-pycolyl)amine (DPA), and a DNA intercalating phenanthrene unit. Their general formula is Cu-DPA-N,N' (where N,N'=1,10-phenanthroline (Phen), dipyridoquinoxaline (DPQ) or dipyridophenazine (DPPZ)). Characterisation was achieved by X-ray crystallography and continuous-wave EPR (cw-EPR), hyperfine sublevel correlation (HYSCORE) and Davies electron-nuclear double resonance (ENDOR) spectroscopies. The presence of the DPA ligand enhances solution stability and facilitates enhanced DNA recognition with apparent binding constants (Kapp ) rising from 105 to 107 m-1 with increasing extent of planar phenanthrene. Cu-DPA-DPPZ, the complex with greatest DNA binding and intercalation effects, recognises the minor groove of guanine-cytosine (G-C) rich sequences. Oxidative DNA damage also occurs in the minor groove and can be inhibited by superoxide and hydroxyl radical trapping agents. The complexes, particularly Cu-DPA-DPPZ, display promising anticancer activity against human pancreatic tumour cells with in-vitro results surpassing the clinical platinum(II) drug oxaliplatin.

1H high field electron-nuclear double resonance spectroscopy at 263 GHz/9.4 T.

We present and discuss the performance of 1H electron-nuclear double resonance (ENDOR) at 263 GHz/9.4 T by employing a prototype, commercial quasi optical spectrometer. Basic instrumental features of the setup are described alongside a comprehensive characterization of the new ENDOR probe head design. The performance of three different ENDOR pulse sequences (Davies, Mims and CP-ENDOR) is evaluated using the 1H BDPA radical. A key feature of 263 GHz spectroscopy - the increase in orientation selectivity in comparison with 94 GHz experiments - is discussed in detail. For this purpose, the resolution of 1H ENDOR spectra at 263 GHz is verified using a representative protein sample containing approximately 15 picomoles of a tyrosyl radical. Davies ENDOR spectra recorded at 5 K reveal previously obscured spectral features, which are interpreted by spectral simulations aided by DFT calculations. Our analysis shows that seven internal proton couplings are detectable for this specific radical if sufficient orientation selectivity is achieved. The results prove the fidelity of 263 GHz experiments in reporting orientation-selected 1H ENDOR spectra and demonstrate that new significant information can be uncovered in complex molecular systems, owing to the enhanced resolution combined with high absolute sensitivity and no compromise in acquisition time.

Polypyridyl-Based Copper Phenanthrene Complexes: A New Type of Stabilized Artificial Chemical Nuclease.

The building of robust and versatile inorganic scaffolds with artificial metallo-nuclease (AMN) activity is an important goal for bioinorganic, biotechnology, and metallodrug research fields. Here, a new type of AMN combining a tris-(2-pyridylmethyl)amine (TPMA) scaffold with the copper(II) N,N'-phenanthrene chemical nuclease core is reported. In designing these complexes, the stabilization and flexibility of TPMA together with the prominent chemical nuclease activity of copper 1,10-phenanthroline (Phen) were targeted. A second aspect was the opportunity to introduce designer phenazine DNA intercalators (e.g., dipyridophenazine; DPPZ) for improved DNA recognition. Five compounds of formula [Cu(TPMA)(N,N')]2+ (where N,N' is 2,2-bipyridine (Bipy), Phen, 1,10-phenanthroline-5,6-dione (PD), dipyridoquinoxaline (DPQ), or dipyridophenazine (DPPZ)) were developed and characterized by X-ray crystallography. Solution stabilities were studied by continuous-wave EPR (cw-EPR), hyperfine sublevel correlation (HYSCORE), and Davies electron-nuclear double resonance (ENDOR) spectroscopies, which demonstrated preferred geometries in which phenanthrene ligands were coordinated to the copper(II) TPMA core. Complexes with Phen, DPQ, and DPPZ ligands possessed enhanced DNA binding activity, with DPQ and DPPZ compounds showing excellent intercalative effects. These complexes are effective AMNs and analysis with spin-trapping scavengers of reactive oxygen species and DNA repair enzymes with glycosylase/endonuclease activity demonstrated a distinctive DNA oxidation activity compared to classical Sigman- and Fenton-type reagents.

ENDOR with band-selective shaped inversion pulses

Electron Nuclear DOuble Resonance (ENDOR) is based on the measurement of nuclear transition frequencies through detection of changes in the polarization of electron transitions. In Davies ENDOR, the initial polarization is generated by a selective microwave inversion pulse. The rectangular inversion pulses typically used are characterized by a relatively low selectivity, with full inversion achieved only for a limited number of spin packets with small resonance offsets. With the introduction of pulse shaping to EPR, the rectangular inversion pulses can be replaced with shaped pulses with increased selectivity. Band-selective inversion pulses are characterized by almost rectangular inversion profiles, leading to full inversion for spin packets with resonance offsets within the pulse excitation bandwidth and leaving spin packets outside the excitation bandwidth largely unaffected. Here, we explore the consequences of using different band-selective amplitude-modulated pulses designed for NMR as the inversion pulse in ENDOR. We find an increased sensitivity for small hyperfine couplings compared to rectangular pulses of the same bandwidth. In echo-detected Davies-type ENDOR, finite Fourier series inversion pulses combine the advantages of increased absolute ENDOR sensitivity of short rectangular inversion pulses and increased sensitivity for small hyperfine couplings of long rectangular inversion pulses. The use of pulses with an almost rectangular frequency-domain profile also allows for increased control of the hyperfine contrast selectivity. At X-band, acquisition of echo transients as a function of radiofrequency and appropriate selection of integration windows during data processing allows efficient separation of contributions from weakly and strongly coupled nuclei in overlapping ENDOR spectra within a single experiment.

Electron Paramagnetic Resonance and Electron Nuclear Double Resonance Study of the Paramagnetic Complexes of Anthraquinone on the Surface of γ-Al2O3

Progress in the synthesis and applications of nanomaterials including nanocatalysts demands a use of precise analytical tools for their surface characterization. Continuous wave (cw) and pulsed electron paramagnetic resonance (EPR) techniques, including electron–nuclear double resonance (ENDOR) have been applied to study paramagnetic complexes formed by adsorption of 9,10-anthraquinone (AQ) as probe molecule by the surface of γ-Al2O3. Up to three different paramagnetic complexes (11-line pattern and two single EPR lines) could be separated in our experiments. Their spectroscopic characteristics are extracted. It is shown that at very high concentration (ca. 10 wt %) of AQ, the obtained EPR signal is close to the single line and can be incorrectly interpreted as due to the EPR signal of AQ itself or due to the lower catalytic activity of the investigated surface. That fact should be taken into account by using AQ as a probe of the surface catalytic activity. Mims and Davies ENDOR experiments confirm the re...

EPR analysis of cyanide complexes of wild-type human neuroglobin and mutants in comparison to horse heart myoglobin

Electron paramagnetic resonance (EPR) data reveal large differences between the ferric (13C-)cyanide complexes of wild-type human neuroglobin (NGB) and its H64Q and F28L point mutants and the cyanide complexes of mammalian myo- and haemoglobin. The point mutations, which involve residues comprising the distal haem pocket in NGB, induce smaller, but still significant changes, related to changes in the stabilization of the cyanide ligand. Furthermore, for the first time, the full 13C hyperfine tensor of the cyanide carbon of cyanide-ligated horse heart myoglobin (hhMb) was determined using Davies ENDOR (electron nuclear double resonance). Disagreement of these experimental data with earlier predictions based on 13C NMR data and a theoretical model reveal significant flaws in the model assumptions. The same ENDOR procedure allowed also partial determination of the corresponding 13C hyperfine tensor of cyanide-ligated NGB and H64QNGB. These 13C parameters differ significantly from those of cyanide-ligated hhMb and challenge our current theoretical understanding of how the haem environment influences the magnetic parameters obtained by EPR and NMR in cyanide-ligated haem proteins.

Enhanced sensitivity of electron-nuclear double resonance (ENDOR) by cross polarisation and relaxation

Electron-nuclear double resonance (ENDOR) is a method of choice to detect magnetic nuclei in the coordination sphere of paramagnetic molecules, but its sensitivity substantially suffers from saturation effects. Recently we introduced a new pulsed ENDOR experiment based on electron-nuclear cross polarisation (CP) transfer. Here we analyse the time evolution of the spin polarization in CP-ENDOR and show that CP combined with inherent fast relaxation leads to enhanced sensitivity as compared to Davies ENDOR.

Increasing sensitivity of pulse EPR experiments using echo train detection schemes

Modern pulse EPR experiments are routinely used to study the structural features of paramagnetic centers. They are usually performed at low temperatures, where relaxation times are long and polarization is high, to achieve a sufficient Signal/Noise Ratio (SNR). However, when working with samples whose amount and/or concentration are limited, sensitivity becomes an issue and therefore measurements may require a significant accumulation time, up to 12h or more. As the detection scheme of practically all pulse EPR sequences is based on the integration of a spin echo – either primary, stimulated or refocused – a considerable increase in SNR can be obtained by replacing the single echo detection scheme by a train of echoes. All these echoes, generated by Carr–Purcell type sequences, are integrated and summed together to improve the SNR. This scheme is commonly used in NMR and here we demonstrate its applicability to a number of frequently used pulse EPR experiments: Echo-Detected EPR, Davies and Mims ENDOR (Electron-Nuclear Double Resonance), DEER (Electron-Electron Double Resonance|) and EDNMR (Electron–Electron Double Resonance (ELDOR)-Detected NMR), which were combined with a Carr–Purcell–Meiboom–Gill (CPMG) type detection scheme at W-band. By collecting the transient signal and integrating a number of refocused echoes, this detection scheme yielded a 1.6–5 folds SNR improvement, depending on the paramagnetic center and the pulse sequence applied. This improvement is achieved while keeping the experimental time constant and it does not introduce signal distortion.

Cross-polarisation edited ENDOR

Electron-nuclear double resonance (ENDOR) is a fundamental technique in electron paramagnetic resonance (EPR) spectroscopy that directly detects hyperfine transitions of nuclei coupled to a paramagnetic centre. Despite its wide use, spin-sensitivity and restricted spectral resolution in powder samples pose limitations of this technique in modern application fields of EPR. In this contribution, we examine the performance of an ENDOR pulse sequence that utilises a preparation scheme different from conventional Davies ENDOR. The scheme is based on electron-nuclear cross-polarisation (eNCP), which requires concomitant microwave (MW) and radio-frequency (RF) irradiation satisfying specific matching conditions between the MW and RF offsets and the hyperfine coupling. Changes in nuclear polarisation generated during eNCP can be detected via a conventional ENDOR read-out sequence consisting of an RF π-pulse followed by EPR-spin echo detection. Using 1H-BDPA as a standard sample, we first examine the CP matching conditions by monitoring the depolarisation of the electron spin magnetisation. Subsequently, so-called CP-edited ENDOR spectra for different matching conditions are reported and analysed based on the provided theoretical description of the time evolution of the spin density matrix during the experiment. The results demonstrate that CP-edited ENDOR provides additional information with respect to the sign of the hyperfine couplings. Furthermore, the sequence is less sensitive to nuclear saturation effects encountered in conventional ENDOR.

HYSCORE and Davies ENDOR study of irradiated ultra high molecular weight polyethylene

Ultra high molecular weight polyethylene (UHMWPE) has been studied with different magnetic resonance techniques to elicit information on the nature and the location of radicals generated during high energy irradiation. Field swept electron paramagnetic resonance, pulsed Davies electron nuclear double resonance and hyperfine sublevel correlation spectroscopic measurements allowed extracting for the first time the full (1) H hyperfine coupling tensors of the most abundant radical, i.e. a secondary alkyl radical and to ascertain the formation of allyl radicals in the first stages of the irradiation process. The (1) H hyperfine coupling tensors are analogous to those reported for single crystal irradiated polyethylene, suggesting that radicals generated in UHMWPE are located in the crystalline region of the polymer.

Synthetic model of the asymmetric [Mn 3 CaO 4 ] cubane core of the oxygen-evolving complex of photosystem II

The laboratory synthesis of the oxygen-evolving complex (OEC) of photosystem II has been the objective of synthetic chemists since the early 1970s. However, the absence of structural information on the OEC has hampered these efforts. Crystallographic reports on photosystem II that have been appearing at ever-improving resolution over the past ten years have finally provided invaluable structural information on the OEC and show that it comprises a [Mn(3)CaO(4)] distorted cubane, to which is attached a fourth, external Mn atom, and the whole unit attached to polypeptides primarily by aspartate and glutamate carboxylate groups. Such a heterometallic Mn/Ca cubane with an additional metal attached to it has been unknown in the literature. This paper reports the laboratory synthesis of such an asymmetric cubane-containing compound with a bound external metal atom, [(1)]. All peripheral ligands are carboxylate or carboxylic acid groups. Variable-temperature magnetic susceptibility data have established 1 to possess an S = 9/2 ground state. EPR spectroscopy confirms this, and the Davies electron nuclear double resonance data reveal similar hyperfine couplings to those of other Mn(IV) species, including the OEC S(2) state. Comparison of the X-ray absorption data with those for the OEC reveal 1 to possess structural parameters that make it a close structural model of the asymmetric-cubane OEC unit. This geometric and electronic structural correspondence opens up a new front in the multidisciplinary study of the properties and function of this important biological unit.

Electronic Structure Description of a [Co(III)3Co(IV)O4] Cluster: A Model for the Paramagnetic Intermediate in Cobalt-Catalyzed Water Oxidation

Multifrequency electron paramagnetic resonace (EPR) spectroscopy and electronic structure calculations were performed on [Co(4)O(4)(C(5)H(5)N)(4)(CH(3)CO(2))(4)](+) (1(+)), a cobalt tetramer with total electron spin S = 1/2 and formal cobalt oxidation states III, III, III, and IV. The cuboidal arrangement of its cobalt and oxygen atoms is similar to that of proposed structures for the molecular cobaltate clusters of the cobalt-phosphate (Co-Pi) water-oxidizing catalyst. The Davies electron-nuclear double resonance (ENDOR) spectrum is well-modeled using a single class of hyperfine-coupled (59)Co nuclei with a modestly strong interaction (principal elements of the hyperfine tensor are equal to [-20(±2), 77(±1), -5(±15)] MHz). Mims (1)H ENDOR spectra of 1(+) with selectively deuterated pyridine ligands confirm that the amount of unpaired spin on the cobalt-bonding partner is significantly reduced from unity. Multifrequency (14)N ESEEM spectra (acquired at 9.5 and 34.0 GHz) indicate that four nearly equivalent nitrogen nuclei are coupled to the electron spin. Cumulatively, our EPR spectroscopic findings indicate that the unpaired spin is delocalized almost equally across the eight core atoms, a finding corroborated by results from DFT calculations. Each octahedrally coordinated cobalt ion is forced into a low-spin electron configuration by the anionic oxo and carboxylato ligands, and a fractional electron hole is localized on each metal center in a Co 3d(xz,yz)-based molecular orbital for this essentially [Co(+3.125)(4)O(4)] system. Comparing the EPR spectrum of 1(+) with that of the catalyst film allows us to draw conclusions about the electronic structure of this water-oxidation catalyst.

Electrical Detection of Coherent Nuclear Spin Oscillations in Phosphorus-Doped Silicon using Pulsed ENDOR

We demonstrate the electrical detection of pulsed X-band electron nuclear double resonance (ENDOR) in phosphorus-doped silicon at 5 K. A pulse sequence analogous to Davies ENDOR in conventional electron spin resonance is used to measure the nuclear spin transition frequencies of the (31)P nuclear spins, where the (31)P electron spins are detected electrically via spin-dependent transitions through Si/SiO(2) interface states, thus not relying on a polarization of the electron spin system. In addition, the electrical detection of coherent nuclear spin oscillations is shown, demonstrating the feasibility to electrically read out the spin states of possible nuclear spin qubits.

Structural Characterization of a High Affinity Mononuclear Site in the Copper(II)-α-Synuclein Complex

Human α-Synuclein (aS), a 140 amino acid protein, is the main constituent of Lewy bodies, the cytoplasmatic deposits found in the brains of Parkinson's disease patients, where it is present in an aggregated, fibrillar form. Recent studies have shown that aS is a metal binding protein. Moreover, heavy metal ions, in particular divalent copper, accelerate the aggregation process of the protein. In this work, we investigated the high affinity binding mode of truncated aS (1-99) (aS99) with Cu(II), in a stoichiometric ratio, to elucidate the residues involved in the binding site and the role of copper ions in the protein oligomerization. We used Electron Paramagnetic Resonance spectroscopy on the Cu(II)-aS99 complex at pH 6.5, performing both multifrequency continuous wave experiments and pulsed experiments at X-band. The comparison of 9.5 and 95 GHz data showed that at this pH only one binding mode is present. To identify the nature of the ligands, we performed Electron Spin Echo Envelope Modulation, Hyperfine Sublevel Correlation Spectroscopy, and pulsed Davies Electron-Nuclear Double Resonance (Davies-ENDOR) experiments. We determined that the EPR parameters are typical of a type-II copper complex, in a slightly distorted square planar geometry. Combining the results from the different pulsed techniques, we obtained that the equatorial coordination is {N(Im), N(-), H(2)O, O}, where N(im) is the imino nitrogen of His50, N(-) a deprotonated amido backbone nitrogen that we attribute to His50, H(2)O an exchangeable water molecule, and O an unidentified oxygen ligand. Moreover, we propose that the free amino terminus (Met1) participates in the complex as an axial ligand. The MXAN analysis of the XAS k-edge absorption data allowed us to independently validate the structural features proposed on the basis of the magnetic parameters of the Cu(II)-aS99 complex and then to further refine the quality of the proposed structural model.

Pulsed EPR and ENDOR on the Peridinin Triplet State Involved in the Photoprotective Mechanism in Peridinin–Chlorophyll a–Proteins

The photoexcited triplet state of the carotenoid peridinin in the peridinin–chlorophyll a–protein (PCP) of the dinoflagellate Heterocapsa pygmaea has been investigated by pulsed electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) spectroscopies. The α- and β-protons hyperfine couplings of the peridinin-conjugated chain have been derived from Davies and Mims ENDOR experiments. The spectroscopic results have been compared to those obtained for the main form of the PCP complex and for the high-salt PCP form from Amphidinium carterae. The EPR features of the peridinin triplet state are very similar in the antenna complexes belonging to the two different dinoflagellate species, proving that the triplet formation pathway and the triplet localization on one specific peridinin per subcluster are common features of different PCP antennas. No significant variation of the hyperfine couplings of the peridinin triplet state has been detected between the main form of the PCP complex from A. carterae and H. pygmaea. The spectroscopic results confirm the close relationship between the Amphidinium PCP and the corresponding Heterocapsa complex at least in terms of mutual arrangement of the chlorophyll a–peridinin pair involved in photoprotection and in terms of conformation of the peridinin-conjugated chain.

Structure and Properties of 9′-cis Neoxanthin Carotenoid Radicals by Electron Paramagnetic Resonance Measurements and Density Functional Theory Calculations: Present in LHC II?

The radical intermediates formed upon catalytic or photooxidation of the carotenoid 9'-cis neoxanthin inside MCM-41 molecular sieves were detected by pulsed Mims and Davies electron nuclear double resonance (ENDOR) spectroscopies and characterized by density functional theory (DFT) calculations. Mims ENDOR spectra (20 K) were simulated using the hyperfine coupling constants predicted by DFT, which showed that a mixture of carotenoid radical cations (Car(+)) and neutral radicals (#Car) is formed. The DFT relative energies of the neutral radicals formed by proton loss from the C5, C5', C9, C9', C13, and C13'-methyl groups of Car(+) showed that #Car(9') is energetically most favorable, while #Car(9), #Car(13), #Car(13'), #Car(5'), and #Car(5) are less favorable for formation by 2.6, 5.0, 5.1, 22.5, and 25.6 kcal/mol. No evidence for formation of #Car(5') and #Car(5) was observed in the EPR spectra, consistent with DFT calculations. The epoxy group at the prime end and the allene bond at the unprime end prevent protons loss at the C5 and C5'-methyl groups by reducing the conjugation so crucial for the neutral radical stability. Previous CV measurements for allene-substituted carotenoids show that once the radical cations are formed, proton loss is rapid. These examined properties and the known crystal structure of the light harvesting complex II (LHC II) suggest the absence of the neutral radicals of 9'-cis neoxanthin available for quenching the excited states of Chl, consistent with its observed nonquenching properties.

The Mn2+−Bicarbonate Complex in a Frozen Solution Revisited by Pulse W-Band ENDOR

The coordination of bicarbonate to Mn (2+) is the simplest model system for the coordination of Mn (2+) to carboxylate residues in a protein. Recently, the structure of such a complex has been investigated by means of X-band pulse EPR (electron paramagnetic resonance) experiments ( Dasgupta, J. ; et al. J. Phys. Chem. B 2006, 110, 5099 ). Based on the EPR results, together with electrochemical titrations, it has been concluded that the Mn (2+) bicarbonate complex consists of two bicarbonate ligands, one of which is monodentate and other bidentate, but only the latter has been observed by the pulsed EPR techniques. The X-band measurements, however, suffer several drawbacks. (i) The zero-field splitting (ZFS) term of the spin Hamiltonian affects the nuclear frequencies. (ii) There are significant contributions from ENDOR (electron nuclear double resonance) lines of the M S not equal +/- (1)/ 2 manifolds. (iii) There are overlapping signals of (23)Na. All these reduce the uniqueness of the data interpretation. Here we present a high-field ENDOR investigation of Mn (2+)/NaH (13)CO 3 in a water/methanol solution that eliminates the above difficulties. Both Davies and Mims ENDOR measurements were carried out. The spectra show that a couple of slightly inequivalent (13)C nuclei are present, with isotropic and anisotropic hyperfine couplings of A iso1 = 1.2 MHz, T perpendicular1 = 0.7 MHz, A iso2 = 1.0 MHz, T perpendicular2 = 0.6 MHz, respectively. The sign of the hyperfine coupling was determined by variable mixing time (VMT) ENDOR measurements. These rather close hyperfine parameters suggest that there are either two distinct, slightly different, carbonate ligands or that there is some distribution in conformation in only one ligand. The distances extracted from T perpendicular1 and T perpendicular2 are consistent with a monodentate binding mode. The monodentate binding mode and the presence of two ligands were further supported by DFT calculations and (1)H ENDOR measurements. Additionally, (23)Na ENDOR resolved at least two types of (23)Na (+) in the Mn (2+)-bicarbonate complex, thus suggesting that the bicarbonate bridges two positively charged metal ions.

Pulsed Electron Nuclear Double Resonance Studies of Carotenoid Oxidation in Cu(II)-Substituted MCM-41 Molecular Sieves

Carotenoid (Car) radical intermediates formed upon catalytic or photooxidation of lutein (I), 7'-apo-7',7'-dicyano-beta-carotene (II), and lycopene (III) inside Cu(II)-MCM-41 molecular sieves were studied by pulsed electron nuclear double resonance (ENDOR) spectroscopies. The Davies and Mims ENDOR spectra (15-20 K) were simulated using the hyperfine coupling constants predicted by density functional theory (DFT) calculations. The DFT calculations revealed that upon chemical oxidation, carotenoid radical cations (Car*+) are formed, whereas carotenoid neutral radicals (#Car*) are produced by proton loss (indicated by #) from the radical cation. This loss is to first order independent of polarity or hydrogen bonding for carotenoids I, II, or III inside Cu(II)-MCM-41 molecular sieves.

A Multi-Frequency Pulse EPR and ENDOR Approach to Study Strongly Coupled Nuclei in Frozen Solutions of High-Spin Ferric Heme Proteins

In spite of the tremendous progress in the field of pulse electron paramagnetic resonance (EPR) in recent years, these techniques have been scarcely used to investigate high-spin (HS) ferric heme proteins. Several technical and spin-system-specific reasons can be identified for this. Additional problems arise when no single crystals of the heme protein are available. In this work, we use the example of a frozen solution of aquometmyoglobin (metMb) to show how a multi-frequency pulse EPR approach can overcome these problems. In particular, the performance of the following pulse EPR techniques are tested: Davies electron nuclear double resonance (ENDOR), hyperfine correlated ENDOR (HYEND), electron-electron double resonance (ELDOR)-detected NMR, and several variants of hyperfine sublevel correlation (HYSCORE) spectroscopy including matched and SMART HYSCORE. The pulse EPR experiments are performed at X-, Q- and W-band microwave frequencies. The advantages and drawbacks of the different methods are discussed in relation to the nuclear interaction that they intend to reveal. The analysis of the spectra is supported by several simulation procedures, which are discussed. This work focuses on the analysis of the hyperfine and nuclear-quadrupole tensors of the strongly coupled nuclei of the first coordination sphere, namely, the directly coordinating heme and histidine nitrogens and the 17O nucleus of the distal water ligand. For the latter, 17O-isotope labeling was used. The accuracy of our results and the spectral resolution are compared in detail to an earlier single-crystal continuous-wave ENDOR study on metMb, and it will be shown how additional information can be obtained from the multi-frequency approach. The current work is therefore prone to become a template for future EPR/ENDOR investigations of HS ferric heme proteins for which no single crystals are available.

Nuclear relaxation effects in Davies ENDOR variants

A recent article by Yang and Hoffman [T.-C. Yang, B.M. Hoffman, J. Magn. Reson., 181 (2006) 280] presents a ‘Davies/Hahn ENDOR multi-sequence’ in which the α and β peaks of an electron-nuclear double resonance (ENDOR) spectrum can be distinguished. This represents one instance of a family of ENDOR sequences which have no initial microwave inversion pulse, and which can reveal information about nuclear relaxation rates and the signs of hyperfine coupling constants. Here we discuss the more general set of such sequences, which we refer to as Saturated Pulsed ENDOR, and show how signal sensitivity can be optimised within the context of this new technique. Through simulations, we compare its performance to other techniques based on Davies ENDOR, and experimentally illustrate its properties using the non-heme Fe enzyme anthranilate dioxygenase AntDO. Finally, we suggest a protocol for extracting both the magnitude and sign of the hyperfine tensor using a combination of ENDOR techniques.