Pulsed Electron Paramagnetic Resonance Experiments Research Articles

Studying Dynamics and Conformational Changes in the Glycine Riboswitch using Electron Paramagnetic Resonance Spectroscopy

Electron Paramagnetic Resonance (EPR) spectroscopy is used to study dynamics and conformational changes in the RNA glycine riboswitch. The dynamic role of the leader-linker interaction within glycine riboswitch conserved sequences is probed through site directed spin labeling and continuous wave EPR. Inter-aptamer and aptamer-expression platform interactions are elucidated through double electron-electron resonance spectroscopy. Incorporation of spin labels is achieved through optimized ligation methodologies allowing synthetically modified RNA to be joined to larger RNA sequences. Expected folding and burial of riboswitch elements will lead to restricted motion of the spin label and, additionally, pulsed EPR experiments yield distance distribution profiles indicating conformational exchange between states in the absence and presence of glycine.

High-frequency EPR study of crude oils

Four different samples of crude oil were studied by means of high-frequency W-band (94 GHz) electron paramagnetic resonance (EPR) spectroscopy with the aim to develop new methods of crude oil quality control. High spectral resolution of W-band allowed to avoid an overlap of spectra contributors. The ratio K between the integral intensity of the low-field EPR component of the vanadyl complexes to that of free radical line was chosen as an attribute of each sample. Using the K-parameters and EPR spectra simulations the crude oil leaking between adjacent horizons is shown. Pulsed EPR experiments allowed detecting free radicals signals only. It is demonstrated that the extracted transverse relaxation time could be used as an additional parameter which characterizes the origin of the crude oil and nature of the oil paramagnetic centers.

A Combined Pulsed Electron Paramagnetic Resonance Spectroscopic and DFT Analysis of the13CO2and13CO Adsorption on the Metal–Organic Framework Cu2.97Zn0.03(btc)2

Cu3(btc)2 (btc = 1,3,5-benzenetricarboxylate), also called HKUST-1, is one of the well-known representatives of the metal–organic framework (MOF) compounds. It exhibits a large surface area and a high pore volume. Due to the coordinatively unsaturated metal centers as preferential adsorption sites, Cu3(btc)2 is particularly interesting for the separation of CO2 and CO in gaseous mixtures. We studied the interactions of 13C-enriched carbon dioxide (13CO2) and carbon monoxide (13CO) with the Cu2+ centers in the zinc-substituted homologue Cu2.97Zn0.03(btc)2 using continuous wave (cw) and pulsed electron paramagnetic resonance (EPR) spectroscopy (Davies or Mims electron nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE)). Upon adsorption of 13CO2 and 13CO, the coordination geometry of the Cu2+ centers changed from square planar to square pyramidal. The cupric ion g-tensor and the 63/65Cu hyperfine coupling tensor ACu show the changes in the ligand field of Cu2+. Moreover, the interaction with the 13C nuclei of the gas molecules is reflected in the isotropic coupling constant AisoC and the dipolar coupling parameter T⊥C which are derived from the 13C hyperfine coupling tensor AC obtained by the pulsed EPR experiments. From the experimentally obtained parameters, we derived a geometrical model for the adsorption of 13CO2 and 13CO at the Cu2+ ions that is consistent with our DFT calculations. The 13CO molecule is found to coordinate linearly at the Cu2+ center via the 13C atom and perpendicular to the CuO4 plane with a Cu–C distance of rCuC = 2.57(10) Å (DFT, 2.42 Å). The 13CO2 molecule is coordinated slightly tilted via the O atom with a Cu–C distance of rCuC = 3.34(10) Å (DFT, 3.27 Å). The Cu–O distance for adsorbed 13CO2 is not directly accessible to EPR measurements but could be estimated from geometrical considerations in the range of rCuO = 2.53–2.73 Å (DFT, 2.39 Å). The results provide detailed insight into the geometry of adsorbed CO2 and CO in porous materials and show the potential of EPR spectroscopy for analyzing adsorption complexes.

Phase cycling with a 240 GHz, free electron laser-powered electron paramagnetic resonance spectrometer

Electron paramagnetic resonance (EPR) powered by a free electron laser (FEL) has been shown to dramatically expand the capabilities of EPR at frequencies above ~100 GHz, where other high-power sources are unavailable. High-power pulses are necessary to achieve fast (<10 ns) spin rotations in order to alleviate the limited excitation bandwidth and time resolution that typically hamper pulsed EPR at these high frequencies. While at these frequencies, an FEL is the only source that provides ~1 kW of power and can be tuned continuously up to frequencies above 1 THz, it has only recently been implemented for one- and two-pulse EPR, and the capabilities of the FEL as an EPR source are still being expanded. This manuscript presents phase cycling of two pulses in an FEL-EPR spectrometer operating at 240 GHz. Given that the FEL, unlike amplifiers, cannot be easily phase-locked to a reference source, we instead apply retrospective data processing to measure the relative phase of each FEL pulse in order to correct the signal phase accordingly. This allows the measured signal to be averaged coherently, and the randomly changing phase of the FEL pulse results in a stochastic phase cycle, which, in the limit of many pulses, efficiently cancels artifacts and improves sensitivity. Further, the relative phase between the first and second pulse, which originates from the difference in path length traversed by each pulse, can be experimentally measured without phase-sensitive detection. We show that the relative phase of the two pulses can be precisely tuned, as well as distinctly switched by a fixed amount, with the insertion of a dielectric material into the quasi-optical path of one of the pulses. Taken together, these techniques offer many of the advantages of arbitrary phase control, and allow application of phase cycling to dramatically enhance signal quality in pulsed EPR experiments utilizing high-power sources that cannot be phase-locked.

A tunable general purpose Q-band resonator for CW and pulse EPR/ENDOR experiments with large sample access and optical excitation

We describe a frequency tunable Q-band cavity (34 GHz) designed for CW and pulse Electron Paramagnetic Resonance (EPR) as well as Electron Nuclear Double Resonance (ENDOR) and Electron Electron Double Resonance (ELDOR) experiments. The TE 011 cylindrical resonator is machined either from brass or from graphite (which is subsequently gold plated), to improve the penetration of the 100 kHz field modulation signal. The (self-supporting) ENDOR coil consists of four 0.8 mm silver posts at 2.67 mm distance from the cavity center axis, penetrating through the plunger heads. It is very robust and immune to mechanical vibrations. The coil is electrically shielded to enable CW ENDOR experiments with high RF power (500 W). The top plunger of the cavity is movable and allows a frequency tuning of ±2 GHz. In our setup the standard operation frequency is 34.0 GHz. The microwaves are coupled into the resonator through an iris in the cylinder wall and matching is accomplished by a sliding short in the coupling waveguide. Optical excitation of the sample is enabled through slits in the cavity wall (transmission ∼60%). The resonator accepts 3 mm o.d. sample tubes. This leads to a favorable sensitivity especially for pulse EPR experiments of low concentration biological samples. The probehead dimensions are compatible with that of Bruker flexline Q-band resonators and it fits perfectly into an Oxford CF935 Helium flow cryostat (4–300 K). It is demonstrated that, due to the relatively large active sample volume (20–30 μl), the described resonator has superior concentration sensitivity as compared to commercial pulse Q-band resonators. The quality factor ( Q L ) of the resonator can be varied between 2600 (critical coupling) and 1300 (over-coupling). The shortest achieved π/2-pulse durations are 20 ns using a 3 W microwave amplifier. ENDOR (RF) π-pulses of 20 μs ( 1H @ 51 MHz) were obtained for a 300 W amplifier and 7 μs using a 2500 W amplifier. Selected applications of the resonator are presented.

Multifrequency Pulsed Electron Paramagnetic Resonance on Metalloproteins

Metalloproteins often contain metal centers that are paramagnetic in some functional state of the protein; hence electron paramagnetic resonance (EPR) spectroscopy can be a powerful tool for studying protein structure and function. Dipolar spectroscopy allows the determination of the dipole-dipole interactions between metal centers in protein complexes, revealing the structural arrangement of different paramagnetic centers at distances of up to 8 nm. Hyperfine spectroscopy can be used to measure the interaction between an unpaired electron spin and nuclear spins within a distance of 0.8 nm; it therefore permits the characterization of the local structure of the paramagnetic center's ligand sphere with very high precision. In this Account, we review our laboratory's recent applications of both dipolar and hyperfine pulsed EPR methods to metalloproteins. We used pulsed dipolar relaxation methods to investigate the complex of cytochrome c and cytochrome c oxidase, a noncovalent protein-protein complex involved in mitochondrial electron-transfer reactions. Hyperfine sublevel correlation spectroscopy (HYSCORE) was used to study the ligand sphere of iron-sulfur clusters in complex I of the mitochondrial respiratory chain and substrate binding to the molybdenum enzyme polysulfide reductase. These examples demonstrate the potential of the two techniques; however, they also highlight the difficulties of data interpretation when several paramagnetic species with overlapping spectra are present in the protein. In such cases, further approaches and data are very useful to enhance the information content. Relaxation filtered hyperfine spectroscopy (REFINE) can be used to separate the individual components of overlapping paramagnetic species on the basis of differences in their longitudinal relaxation rates; it is applicable to any kind of pulsed hyperfine or dipolar spectroscopy. Here, we show that the spectra of the iron-sulfur clusters in complex I can be separated by this method, allowing us to obtain hyperfine (and dipolar) information from the individual species. Furthermore, performing pulsed EPR experiments at different magnetic fields is another important tool to disentangle the spectral components in such complex systems. Despite the fact that high magnetic fields do not usually lead to better spectral separation for metal centers, they provide additional information about the relative orientation of different paramagnetic centers. Our high-field EPR studies on cytochrome c oxidase reveal essential information regarding the structural arrangement of the binuclear Cu(A) center with respect to both the manganese ion within the enzyme and the cytochrome in the protein-protein complex with cytochrome c.

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.

Time-optimal synthesis of unitary transformations in a coupled fast and slow qubit system

In this paper, we study time-optimal control problems related to a system of two coupled qubits where the time scales involved in performing unitary transformations on each qubit are significantly different. In particular, we address the case where unitary transformations produced by evolutions of the coupling take a much longer time as compared to the time required to produce unitary transformations on the first qubit, but a much shorter time as compared to the time to produce unitary transformations on the second qubit. We present a canonical decomposition of SU(4) in terms of the subgroup $\text{SU}(2)\ifmmode\times\else\texttimes\fi{}\text{SU}(2)\ifmmode\times\else\texttimes\fi{}\text{U}(1)$, which is natural in understanding the time-optimal control problem of such a coupled qubit system with significantly different time scales. A typical setting involves dynamics of a coupled electron-nuclear spin system in pulsed electron paramagnetic resonance experiments at high fields. Using the proposed canonical decomposition, we give time-optimal control algorithms to synthesize various unitary transformations of interest in coherent spectroscopy and quantum-information processing.

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.

Pulsed Electron Paramagnetic Resonance Experiments Identify the Paramagnetic Intermediates in the Pyruvate Ferredoxin Oxidoreductase Catalytic Cycle

Pyruvate ferredoxin oxidoreductase (PFOR) is central to the anaerobic metabolism of many bacteria and amitochondriate eukaryotes. PFOR contains thiamine pyrophosphate (TPP) and three [4Fe-4S] clusters, which link pyruvate oxidation to reduction of ferredoxin. In the PFOR reaction, TPP reacts with pyruvate to form lactyl-TPP, which undergoes decarboxylation to form a hydroxyethyl-TPP (HE-TPP) intermediate. One electron is then transferred from HE-TPP to one of the three [4Fe-4S] clusters to form an HE-TPP radical and a [4Fe-4S]1+ intermediate. Pulsed EPR methods have been used to measure the distance between the HE-TPP radical and the [4Fe-4S]1+ cluster to which it is coupled. Computational analysis including the PFOR crystal structure and the spin distribution in the HE-TPP radical and in the reduced [4Fe-4S] cluster demonstrates that the distance between the HE-TPP radical and the medial cluster B matches the experimentally determined dipolar interaction, while one of the other two clusters is too close and the other is too far away. These results clearly demonstrate that it is the medial cluster (cluster B) that is reduced. Thus, rapid electron transfer occurs through the electron-transfer chain, which leaves an oxidized proximal cluster poised to accept an electron from the HE-TPP radical in the subsequent reaction step.

26.5–40 GHz Ka-band pulsed EPR spectrometer

The design and performance of a new homebuilt Ka-band pulsed electron paramagnetic resonance (EPR) spectrometer is described. The spectrometer is unique in that it is broadband and allows experiments to be performed at any operational frequency within the range from 26.5 to 40 GHz. The high-power microwave amplifier employed in this spectrometer allowed us to use resonators with low quality factor and to achieve a minimum inversion microwave pulse duration of 11 ns. As a result, this spectrometer can be used for all types of pulsed EPR experiments, including electron spin echo envelope modulation (ESEEM), electron-nuclear double resonance (ENDOR), and electron-electron double resonance (ELDOR) with a difference between the pumping and observation frequencies of up to several hundred megahertz. The timing is controlled by an arbitrary waveform generator with 12 digital outputs and 1 ns time resolution. The spectrometer control/data acquisition software is home-written and allows an easy implementation of any one- or two-dimensional experiment using sweep definition tables. © 2006 Wiley Periodicals, Inc. Concepts Magn Reson Part B (Magn Reson Engineering) 29B: 125–136, 2006

Synthesis of Dendritic Metalloporphyrins with Distal H‐Bond Donors as Model Systems for Hemoglobin

AbstractWe report the synthesis of the first‐ (G1) and second‐generation (G2) dendritic FeII porphyrins 1⋅Fe–4⋅Fe (G1) and 6⋅Fe (G2) bearing distal H‐bond donors ideally positioned for stabilization of FeIIO2 adducts by H‐bonding (Fig. 1). A first approach towards the construction of these novel biomimetic systems failed unexpectedly: the Suzuki cross‐coupling between appropriately functionalized ZnII porphyrins and ortho‐ethynylated aryl derivatives, serving as anchors for the distal H‐bond donor moieties, was unsuccessful (Schemes 1, 3, and 5), presumably due to steric hindrance resulting from unfavorable coordination of the ethynyl residue to the Pd species in the catalytic cycle (Scheme 6). The target molecules were finally prepared by a route in which the ortho‐ethynylated meso‐aryl ring is introduced during porphyrin construction in a mixed condensation involving the two dipyrrylmethanes 33 and 34, and aldehyde 36 (Schemes 7 and 8). Following attachment of the dendrons (Scheme 11), the distal H‐bond donors were introduced by Sonogashira cross‐coupling (Scheme 12), and subsequent metallation afforded the dendritic FeII porphyrins 1⋅Fe–6⋅Fe. 1H‐NMR Spectroscopy proved the location of the H‐bond donor moiety atop the porphyrin surface, and X‐ray crystal‐structure analysis of model system 45 (Fig. 2) revealed that this moiety would not sterically interfere with gas binding. With 1,2‐dimethyl‐1H‐imidazole (DiMeIm) as ligand, the dendritic FeII porphyrins formed five‐coordinate high‐spin complexes (Figs. 3 and 4) and addition of CO led reversibly to the corresponding stable six‐coordinate gas complexes (Fig. 6). Oxygenation, however, did not result in defined FeIIO2 complexes as rapid decomposition to FeIII species took place immediately, even in the case of the G2 dendrimer 6⋅Fe(DiMeIm) (Fig. 7). In contrast, stable gas adducts are formed between dendritic CoII porphyrins and O2 in the presence of DiMeIm as axial ligand, as revealed by electron paramagnetic resonance (EPR). The possible stabilization of these complexes through H‐bonding involving the distal ligand is currently under investigation in multidimensional and multifrequency pulse EPR experiments.

Pulse programmer for electron paramagnetic resonance spectroscopy

AbstractThe design of a new pulse programmer is presented. It is based on an application‐specific integrated circuit (ASIC) and designed to fulfill the requirements of pulse electron paramagnetic resonance (EPR). The pulse EPR ASIC provides a high time resolution (2 ns) and a high channel density (8 channels per chip). The pulse sequences are controlled by a digital signal processor (DSP). The sequence processing is solved in a general way: the pulse program, written in a high‐level Pulse Programming Language (PPL), is interpreted by the DSP, which then calculates a bit image, loads it to the ASICs, and controls the sequence flow. The approach allows any sequence to be easily programmed, and the pulse EPR experiments can be carried out in real time on a repetition rate of up to 2 kHz. The new pulse programmer provides the same or even a higher time resolution and channel density than general‐purpose commercial programmers, which are in use in home‐built EPR spectrometers. At the same time, the device has a shorter reprogramming time than these programmers and can compete, in particular for 2D experiments, with the existing pulse EPR‐oriented commercial systems. In comparison with commercial analogs the software provides the users with a more flexible control over the sequence programming. The capacity of the programmer for work is demonstrated by three‐pulse ESEEM and HYSCORE experiments. © 2004 Wiley Periodicals, Inc. Concepts Magn Reson Part B (Magn Reson Engineering) 21B: 1–10, 2004.

Applications of pi-photon-induced transparency in two-frequency pulse electron paramagnetic resonance experiments.

An approach to pulse electron paramagnetic resonance (EPR) experiments which are based on two different resonance fields is introduced. Instead of using two microwave (mw) sources or a magnetic field jump, bichromatic pulses consisting of a transverse microwave field with frequency omega(mw) and a longitudinal radio frequency field with frequency omega(rf) are employed. Such bichromatic pulses excite a number of multiple photon transitions at frequencies omega(mw)+komega(rf) (k in Z). The pi-photon-induced transparency phenomenon is used to select the required transitions. This approach is used in the stimulated soft electron spin echo envelope modulation and the four-pulse double electron-electron resonance experiments. The results obtained using the bichromatic pulse approach are in agreement with those obtained with the standard pulse EPR techniques. It is shown that applying bichromatic pulses is straightforward and advantageous in several respects.

A new step in high-frequency EPR of defects in semiconductors

An electron paramagnetic resonance (EPR) spectrometer is described which allows for continuous-wave and pulsed EPR experiments at 275GHz (wavelength 1.1mm). The related magnetic field of 9.9T for g∼2 is supplied by a superconducting solenoid. The microwave bridge employs quasi-optical as well as conventional waveguide components. A cylindrical, single-mode cavity provides a high filling factor and a high sensitivity for EPR detection. Even with the available microwave power of 1mW incident at the cavity a high microwave magnetic field B1 is obtained of about 0.1mT which permits π/2-pulses as short as 100ns. The performance of the spectrometer is illustrated with the help of spectra taken with several samples.

A continuous-wave and pulsed electron spin resonance spectrometer operating at 275 GHz

An electron paramagnetic resonance (EPR) spectrometer is described which allows for continuous-wave and pulsed EPR experiments at 275 GHz (wavelength 1.1 mm). The related magnetic field of 9.9 T for g∼2 is supplied by a superconducting solenoid. The microwave bridge employs quasi-optical as well as conventional waveguide components. A cylindrical, single-mode cavity provides a high filling factor and a high sensitivity for EPR detection. Even with the available microwave power of 1 mW incident at the cavity a high microwave magnetic field B 1 is obtained of about 0.1 mT which permits π/2-pulses as short as 100 ns. The performance of the spectrometer is illustrated with the help of spectra taken with several samples.

High-field/high-frequency EPR spectrometer operating in pulsed and continuous-wave mode at 180 GHz

A novel electron paramagnetic resonance (EPR) spectrometer is reported, which has been developed to allow pulsed EPR experiments with high sensitivity and time resolution at a microwave (MW) frequency of 180 GHz (G-band) and wavelengths of approximately 1.6 mm. This corresponds to a magnetic field of about 6.4 T forg ≈ 2 signals. The “hybrid” system architecture combines components of quasioptical as well as conventional MW techniques, making it possible to achieve excellent spectrometer performance with respect to sensitivity and time resolution. Quasioptical MW components have been used to design an MW circulator allowing high sensitivity and low bias operation in the reflection mode. A miniaturized, closed-type cylindrical cavity provides a high sample filling factor and an adequate MW field strength (B1) enhancement and thus permits reasonably short MW pulses (60 ns for a π/2 pulse) even with a moderate MW input power (15 mW at the cavity). Commercial quartz capillaries (up to 0.5 mm internal diameter) can be used as sample holders for a broad range of applications.

EPR investigation of Cu2+-substituted photosynthetic bacterial reaction centers: evidence for histidine ligation at the surface metal site.

The coordination environments of two distinct metal sites on the bacterial photosynthetic reaction center (RC) protein were probed with pulsed electron paramagnetic resonance (EPR) spectroscopy. For these studies, Cu2+ was bound specifically to a surface site on native Fe2+-containing RCs from Rhodobacter sphaeroides R-26 and to the native non-heme Fe site in biochemically Fe-removed RCs. The cw and pulsed EPR results clearly indicate two spectroscopically different Cu2+ environments. In the dark, the RCs with Cu2+ bound to the surface site exhibit an axially symmetric EPR spectrum with g(parallel) = 2.24, A(parallel) = 160 G, g(perpendicular) = 2.06, whereas the values g(parallel) = 2.31, A(parallel) = 143 G, and g(perpendicular) = 2.07 were observed when Cu(2+) was substituted in the Fe site. Examination of the light-induced spectral changes indicate that the surface Cu2+ is at least 23 A removed from the primary donor (P+) and reduced quinone acceptor (QA-). Electron spin-echo envelope modulation (ESEEM) spectra of these Cu-RC proteins have been obtained and provide the first direct solution structural information about the ligands in the surface metal site. From these pulsed EPR experiments, modulations were observed that are consistent with multiple weakly hyperfine coupled 14N nuclei in close proximity to Cu2+, indicating that two or more histidines ligate the Cu2+ at the surface site. Thus, metal and EPR analyses confirm that we have developed reliable methods for stoichiometrically and specifically binding Cu2+ to a surface site that is distinct from the well characterized Fe site and support the view that Cu2+ is bound at or near the Zn site that modulates electron transfer between the quinones QA and QB (QA-QB --> QAQB-) (Utschig, L. M., Ohigashi, Y., Thurnauer, M. C., and Tiede, D. M (1998) Biochemistry 37, 8278-8281) and proton uptake by QB- (Paddock, M. L., Graige, M. S., Feher, G., and Okamura, M. Y. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 6183-6188). Detailed EPR spectroscopic characterization of these Cu2+-RCs will provide a means to investigate the role of local protein environments in modulating electron and proton transfer.

Dark magnetic resonance in an electron-nuclear spin system

Dark magnetic resonance in the solid state is observed and is shown to be analogous to electromagnetically induced transparency in coherent optics. The basis of the two effects is coherent population trapping, which can conveniently be described by the product operator formalism. It is demonstrated that pulse electron paramagnetic resonance experiments on electron-nuclear spin systems provide a simple means for studying the physics of these types of coherence phenomena.

Magic-angle spinning electron paramagnetic resonance spectroscopy

Pulsed electron paramagnetic resonance (EPR) has been combined magic-angle spinning (MAS) to extract highly-resolved isotropic EPR signals from anisotropic powder spectra. With a self-built probe head, pulsed EPR experiments are performed during sample rotation with a spinning speed of almost 17 kHz. Analogous to the ultra-slow MAS experiment in NMR, in the MAS-EPR experiment transverse magnetization evolves at selected rotor positions, leading to an averaging of anisotropic contributions. The MAS-EPR spectrum of E 1 ′ centers in γ-irradiated silica has been recorded, where averaging of the anisotropic g-factor leads to an isontropic resonance line of 1 MHz spectral width, comparable to EPR lines in low viscous liquids.