Two-pulse Electron Spin Echo Envelope Modulation Research Articles

Hyperfine spectroscopy in a quantum-limited spectrometer.

We report measurements of electron-spin-echo envelope modulation (ESEEM) performed at millikelvin temperatures in a custom-built high-sensitivity spectrometer based on superconducting micro-resonators. The high quality factor and small mode volume (down to 0.2 pL) of the resonator allow us to probe a small number of spins, down to . We measure two-pulse ESEEM on two systems: erbium ions coupled to nuclei in a natural-abundance crystal and bismuth donors coupled to residual nuclei in a silicon substrate that was isotopically enriched in the isotope. We also measure three- and five-pulse ESEEM for the bismuth donors in silicon. Quantitative agreement is obtained for both the hyperfine coupling strength of proximal nuclei and the nuclear-spin concentration.

Relaxation and modulation interference effects in two-pulse electron spin echo envelope modulation (ESEEM)

Two-pulse electron spin echo envelope modulation (ESEEM) line widths are influenced by transverse electron spin relaxation, which is in turn induced by local field fluctuations. Simultaneous analysis of the decays of the unmodulated and modulated parts of the ESEEM signal provides deeper insight into the relaxation of a spin system consisting of an electron spin 1/2 coupled to NI nuclei with spin 1/2. Standard two-pulse ESEEM formulas either do not account for relaxation or assume uniform relaxation for all lines. In general, the relaxation rates on allowed and forbidden transitions may not be the same. Experimental results obtained on a single crystal of Cu(II)-doped l-histidine suggest that such a difference exists. Theoretical considerations show that in such a case the product rule for two-pulse ESEEM does not extend to expressions including relaxation. Product rules in general do not properly account for relaxation in three-pulse ESEEM and HYSCORE experiments. Decay of the apparently non-oscillatory part of the two-pulse echo may be strongly affected by modulation interference. Such interference of difference frequencies of matrix nuclei may cause a rather flat initial feature, which was previously attributed solely to non-exponential phase relaxation of electron spin transitions due to spin diffusion of the matrix nuclei. In addition, the sometimes observed drastic initial decay of the time domain signal is related to modulation interference of multiple-quantum coherences that arise from a strong cross-suppression effect.

Pulse Electron Spin Resonance Studies of H and D Atoms in Impurity-Helium Solids

We have performed measurements of the two-pulse ESEEM (electron spin echo envelope modulation) spectra of H and D atoms within impurity-helium solids. The local environments of the atoms were determined from the modulation of the ESEEM signal by neighboring D2 and HD molecules. We have measured changes in the atom environments due to coalescence of the nanoclusters within the impurity-helium solids and due to the tunneling exchange chemical reaction D+HD→H+D2.

Interaction of the substrate radical and the 5'-deoxyadenosine-5'-methyl group in vitamin B(12) coenzyme-dependent ethanolamine deaminase.

The distance and relative orientation of the C5' methyl group of 5'-deoxyadenosine and the substrate radical in vitamin B(12) coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized by using X-band two-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy in the disordered solid state. The (S)-2-aminopropanol-generated substrate radical catalytic intermediate was prepared by cryotrapping steady-state mixtures of enzyme in which catalytically exchangeable hydrogen sites in the active site had been labeled by previous turnover on (2)H(4)-ethanolamine. Simulation of the time- and frequency-domain ESEEM requires two types of coupled (2)H. The strongly coupled (2)H has an effective dipole distance (r(eff)) of 2.2 A, and isotropic coupling constant (A(iso)) of -0.35 MHz. The weakly coupled (2)H has r(eff) = 3.8 A and A(iso) = 0 MHz. The best (2)H ESEEM time- and frequency-domain simulations are achieved with a model in which the hyperfine couplings arise from one strongly coupled hydrogen site and two equivalent weakly coupled hydrogen sites located on the C5' methyl group of 5'-deoxyadenosine. This model indicates that the unpaired electron on C1 of the substrate radical and C5' are separated by 3.2 A and are thus at closest contact. The close proximity of C1 and C5' indicates that C5' of the 5'-deoxyadenosyl moiety directly mediates radical migration between cobalt in cobalamin and the substrate/product site over a distance of 5-7 A in the active site of ethanolamine deaminase.

Continuous Wave and Pulse EPR and ENDOR Study of Oxygenated Cobalt(II) Heme Model Systems

Continuous wave (CW) and pulse electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR) techniques were used to study frozen solutions of (oxyCo)TPP(L) (TPP = tetraphenylporphyrin, L = pyridine, 1-methylimidazole). By using a combination of CW EPR at X-, Q-, and W-band and Davies-ENDOR at Q-band, the g and cobalt hyperfine matrices and their corresponding principal axes were, for the first time, determined in detail. The variation of the g values as a function of temperature was followed by Q-band CW EPR. The existing ambiguity about the assignment of one of the g principal axes to the O−O bound direction was analyzed in detail. By studying the different proton interactions with Mims-ENDOR at X-band, a considerable solvent effect was found. With hyperfine sublevel correlation (HYSCORE) at X-band and two-pulse ESEEM (electron spin-echo envelope modulation) at S-band, the hyperfine and nuclear quadrupole interactions of the nitrogen nuclei of the axial ligand and the TPP ligand w...

Inhibitory copper binding site on the spinach cytochrome b6f complex: implications for Qo site catalysis.

The isolated cytochrome (cyt) b(6)f complex from spinach is inhibited by Cu(2+) with a K(D) of about 1 microM at pH 7.6 in the presence of 1.6 microM decyl-plastoquinol (C(10)-PQH(2)) as a substrate. Inhibition was competitive with respect to C(10)-PQH(2) but noncompetitive with respect to horse heart cyt c or plastocyanin (PC). Inhibition was also pH-sensitive, with an apparent pK at about 7, above which inhibition was stronger, suggesting that binding occurred at or near a protonatable amino acid residue. Equilibrium binding titrations revealed ca. 1.4 tight Cu(2+) binding sites with a K(D) of about 0.5 microM and multiple (>8) weak (K(D) > 50 microM) binding sites per complex. Pulsed electron paramagnetic resonance (EPR) techniques were used to identify probable binding sites for inhibitory Cu(2+). A distinct enhancement of the relaxation time constant for the EPR signal from bound Cu(2+) was observed when the cyt f was paramagnetic. The magnitude and temperature-dependence of this relaxation enhancement were consistent with a dipole interaction between Cu(2+) and the cyt f (Fe(3+)) heme at a distance of between 30 and 54 A, depending upon the relative orientations of Cu(2+) and cyt f heme g-tensors. Two-pulse electron spin-echo envelope modulation (ESEEM) and 4-pulse 2-dimensional hyperfine sublevel correlation (2D HYSCORE) measurements of Cu(2+) bound to isolated cyt b(6)f complex indicated the presence of a weakly coupled nitrogen nucleus. The nuclear quadrupole interaction (NQI) and the hyperfine interaction (HFI) parameters identified one Cu(2+) ligand as an imidazole nitrogen of a His residue, and electron-nuclear double resonance (ENDOR) confirmed the presence of a directly coordinated nitrogen. A model of the 3-dimensional structure of the cytochrome b(6)f complex was constructed on the basis of sequences and structural similarities with the mitochondrial cyt bc(1) complex, for which X-ray structures have been solved. This model indicated three possible His residues as ligands to inhibitory Cu(2+). Two of these are located on the "Rieske" iron-sulfur protein protein (ISP) while the third is found on the cyt f protein. None of these potential ligands appear to interact directly with the quinol oxidase (Q(o)) binding pocket. A model is thus proposed wherein Cu(2+) interferes with the interaction of the ISP protein with the Q(o) site, preventing the binding and subsequent oxidation of plastoquinonol. Implications for the involvement of ISP "domain movement" in Q(o) site catalysis are discussed.

Generation and transfer of coherence in electron-nuclear spin systems by non-ideal microwave pulses

A quantum mechanical treatment based on a suitably truncated Hamiltonian is developed for the evolution during microwave (MW) irradiation of an S = 1/2, I = 1/2 electron–nuclear spin system with anisotropic hyperfine coupling. It is shown that there is a close analogy between this problem and the well understood phenomena in heteronuclear spin systems with resolved J couplings during double irradiation (L. Müller and R. R. Ernst, 1979, Molec. Phys., 38, 963). A general expression is derived for the MW field strength necessary to establish the modified Hartmann–Hahn condition where electron and nuclear spin states mix completely and maximum nuclear coherence can be generated. The theory is then applied to the discussion of hyperfine decoupling and coherence generation from Boltzmann equilibrium by a MW pulse of arbitrary field strength and duration. It is demonstrated that the build-up of nuclear coherence during such a pulse is slow and oscillatory, and that maximum nuclear coherence is achieved at nominal flip angles considerably larger than 2 π. The creation of both nuclear coherence and coherence on forbidden electron transitions is found to be optimum at the modified Hartmann–Hahn condition, and not to be limited by the modulation depth parameter known from electron spin echo envelope modulation (ESEEM) theory. The theoretical results are used to interpret a number of recent experimental observations that could not be understood by existing theories, and to propose an alternative explanation for nuclear modulations observed in experiments on bacterial photosystems. Effects in systems with a nuclear spin I = 1 are discussed taking into account nuclear quadrupole interactions. It is demonstrated both by numerical simulations and experiments that the well established two-pulse ESEEM experiment can be optimized by using pulses with matched microwave field strength and nominal flip angles larger than 2 π.

Magnetic Field (g-Value) Dependence of Proton Hyperfine Couplings Obtained from ESEEM Measurements: Determination of the Orientation of the Magnetic Axes of Model Heme Complexes in Glassy Media

Electron spin echo envelope modulation (ESEEM) studies were utilized to characterize the coupling between protons of axially bound pyrazole ligands (PzH) and the unpaired electron of low-spin tetraphenylporphyrinatoiron(III) chloride. Samples were prepared in mixed-solvent glasses to maximize the resolution of the electron paramagnetic resonance (EPR) signals. X-band two-pulse ESEEM experiments at 4.2 K in deuterated solvent glasses demonstrated that this coupling results in 0.2−0.7 MHz shifts of the να + νβ proton sum combination peak from twice the Larmor frequency. These shifts have been investigated across most of the EPR absorption spectrum of [TPPFe(PzH)2]+Cl-. Two-pulse ESEEM spectra were simulated at different magnetic field positions. Combination peaks were observed from both distant protons (DP) (β-pyrrole, ortho-phenyl, and β-pyrazole) and near protons (NP) (α-H of the pyrazole ligands). For the simulations, the orientation of the nearest four protons of the pyrazole ligands with respect to the...

Characterization of Histidine Coordination in VO2+-Substituted D-Xylose Isomerase by Orientationally-Selected Electron Spin-Echo Envelope Modulation Spectroscopy

An orientationally-selected electron spin-echo envelope modulation (ESEEM) spectroscopy investigation was performed on VO{sup 2+} introduced into the high-affinity metal-binding site of D-xylose isomerase. The ESEEM spectra clearly reveal the presence of nitrogen ligands with hyperfine coupling A{sup N} {approx} 6 MHz. Detailed analysis includes first- and second-order treatment of the nitrogen basic and combination harmonics in two-pulse ESEEM spectra of the g{sub {parallel}} and g{sub {perpendicular}} components. Complete determination of the hyperfine and quadrupole tensor indicates equatorial coordination of the imine nitrogen of the histidine residue. The presence of Cd{sup 2+} ion in the second, low-affinity metal-binding site does not affect the nitrogen couplings. The protons surrounding the VO{sup 2+} ion have been examined via the proton sum combinations in four-pulse ESEEM. They demonstrate the contribution of two protons probably belonging to the histidine ligand. These investigations strongly support the further application of VO{sup 2+} as a spin probe in conjunction with ESEEM spectroscopy for detailed investigation of nitrogen ligands in the active metal sites of proteins. 41 refs., 8 figs., 2 tabs.

Structural characterization of mononuclear Cu(II) and its nitrite complex in the active site of Carcinus maenas hemocyanin

The preparation of a mononuclear Cu(II) derivative of Carcinus maenas hemocyanin (Cu(II)-Hc) and a nitrite complex of the derivative (Cu(II)-Hc-NO2-) are described. Several techniques have been used in their characterization, including X-ray absorption, continuous wave (cw) EPR, and electron spin-echo envelope modulation (ESEEM) spectroscopies. EXAFS results for Cu(II)-Hc indicate the presence of three ligands at 1.99 +/- 0.01 A and a fourth one at 2.26 +/- 0.01 A from the copper. The same coordination number and very similar bond lengths were obtained for Cu(II)-Hc-NO2-. On the basis of simulations of three-pulse ESEEM spectra, three equivalent imidazole nitrogens coupled to Cu(II) were identified in Cu(II)-Hc. Upon the binding of nitrite, a decrease in the hyperfine interaction for two of the three imidazole nitrogens was observed by ESSEM. Further, the results of a two-pulse ESEEM experiment are consistent with the assignment of the protons of a water ligand to Cu(II), which is displaced when nitrite is added. An analysis of X-ray absorption K-edge spectra suggests a coordination geometry intermediate between square-planar and tetrahedral for the metal centers in Cu(II)-Hc and Cu(II)-Hc-NO2-, in agreement with the g and ACu values determined by cw-EPR. On the basis of these results, an equivalent structure is suggested for Cu(II)-Hc-NO2- and the Cu(II) site in green half-methemocyanin, a partially oxidized binuclear derivative formed in the reaction of the native protein with nitrite.

Observation of spin-locked electron spin echo envelope modulation

Electron spin echo envelope modulation (ESEEM) is observed in a coal sample utilizing a two-pulse sequence originally proposed by Hartmann and Hahn to generate a ‘spin-locked echo’. It is experimentally shown that this spin-locked ESEEM (SLESEEM) is similar to three-pulse ESEEM rather than conventional two-pulse ESEEM with respect to the presence or absence of the sum and the difference frequencies, and the echo decay time. Besides, no phase-cycling is required for the SLESEEM experiment. A qualitative explanation of the new phenomena is suggested.

Sum Combination Harmonics in Four-Pulse ESEEM Spectra. Study of the Ligand Geometry in Aqua-Vanadyl Complexes in Polycrystalline and Glass Matrices

Structural differences in the vanadyl pentaaqua complex in two different matrices, a frozen glassy water solution and a polycrystalline Tutton salt, were investigated using the recently designed one dimensional four-pulse electron spin-echo envelope modulation (ESEEM) experiment. The four-pulse ESEEM spectrum consists of the basic nuclear frequencies, να and νβ, of the ligands′ protons along with their sum combination harmonics, (να + νβ). This particular example demonstrates the potential of this new experiment which lies in the generation of highly resolved combination harmonics from which information regarding the hyperfine and quadrupole interactions can be derived. While the combination lines in the two-pulse ESEEM spectra of the polycrystalline sample suffered from low resolution due to a relatively short T2, highly resolved peaks were observed in the four-pulse ESEEM spectra since in this experiment T1 dominates the echo decay rather than T2. Orientation-selective four-pulse ESEEM experiments were performed under conditions that optimize the intensities of the sum combination harmonics. Aqua complexes with H2O and D2O were studied and clear differences in the Fourier transform ESEEM spectra of the two matrices, not detected previously by electron-nuclear double-resonance spectroscopy or two-pulse ESEEM, were evident. The analysis of the spectra was done by simulating the FT-ESEEM spectra of both the H2O and the D2O complexes. For this purpose, an analytical expression describing the four-pulse ESEEM for I = 1 was derived, taking into account the 2H quadrupole interaction as a first-order correction to the nuclear frequencies. The simulations showed that the positions and orientations of the ligands in VO2+(H2O)5 and VO2+(D2O)5 are somewhat different in the frozen glassy solution and in the crystalline matrix of the Tutton salt.

The manganese center of oxygen-evolving and calcium-depleted photosystem II: a pulsed EPR spectroscopy study

The environment of the multi-manganese center in the O2-evolving complex (OEC) of plant photosystem II (PS II) under conditions of Ca2+ depletion has been probed using pulsed electron paramagnetic resonance (EPR) spectroscopy, and the following results are reported: (1) In Ca(2+)-depleted PS II membranes treated with the chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), the modified Mn EPR signal arising from the OEC in the S2 state and the split EPR signal from the S3 state could be detected in the absorption mode by recording the amplitude of a two-pulse echo as a function of the external magnetic field. The formation of the S3 signal (g approximately 2.004; delta Hpp = 164 G) is not accompanied by the disappearance of the Mn EPR signal, although the signal becomes difficult to detect in CW EPR. This result supports the previous interpretation of the split S3 EPR signal as arising from the interaction of an organic radical with the Mn cluster [Boussac, A., Zimmermann, J. L., Rutherford, A. W., & Lavergne, J. (1990) Nature 347, 303-306]. (2) The two-pulse electron spin echo envelope modulation (ESEEM) spectra of the S2 state formed in Ca(2+)-depleted PS II membranes obtained from 14N- and 15N-labeled material are different. This indicates that nitrogen nuclei from nitrogen-containing protein residues are coupled to the Mn center in the S2 state of the inhibited enzyme. In addition, comparison with the two-pulse ESEEM data obtained for the S2 state in the untreated enzyme suggests that the coupling may be altered by the Ca2+ depletion and/or EGTA treatment. (3) The treatment of Ca(2+)-depleted PS II membranes with sodium pyrophosphate also induced a stable S2 state characterized by a modified multiline EPR signal that is similar to that obtained in EGTA-treated PS II membranes. Comparison of the ESEEM data obtained for the pyrophosphate and 14N and 15N samples treated with EGTA suggests that the modification induced by the EGTA treatment is accompanied by the binding of (an) EGTA molecule(s) to or near the Mn center. (4) ESEEM data obtained for the S3 state formed in the pyrophosphate or EGTA-treated enzyme are quite similar to those obtained for the corresponding S2 state. The data are also compared with ESEEM data obtained on oxidized 4(5)-methylimidazole obtained by UV irradiation. These results are discussed with respect to the current assignment of the S3 radical as arising from oxidation of a histidine residue.

Novel analytical treatments of electron spin-echo envelope modulation with short and extended pulses

The analytical description of two-pulse electron spin-echo envelope modulation experiments is extended by the derivation of two generalized expressions for the echo amplitude. At first, the effect of a variable position of the observer window is computed for delta function pulses. A new type of peak suppression effect, the so-called “observer blind spots,” is discussed. Second, a novel procedure for handling pulses of extended length is introduced. It is based on a pulse representation in terms of a sequence of pulse slices and leads to the first analytical expression that correctly describes an ESEEM experiment performed with pulses of any length, provided the EPR linewidth is much larger than the inverse pulse length. The predicted features are verified experimentally, in particular, the formation of blind spots and the influence of the position and the duration of the observer window.