Zero field splitting fluctuations induced phase relaxation of Gd3+ in frozen solutions at cryogenic temperatures.

Distance measurements using double electron-electron resonance (DEER) and Gd(3+) chelates for spin labels (GdSL) have been shown to be an attractive alternative to nitroxide spin labels at W-band (95GHz). The maximal distance that can be accessed by DEER measurements and the sensitivity of such measurements strongly depends on the phase relaxation of Gd(3+) chelates in frozen, glassy solutions. In this work, we explore the phase relaxation of Gd(3+)-DOTA as a representative of GdSL in temperature and concentration ranges typically used for W-band DEER measurements. We observed that in addition to the usual mechanisms of phase relaxation known for nitroxide based spin labels, GdSL are subjected to an additional phase relaxation mechanism that features an increase in the relaxation rate from the center to the periphery of the EPR spectrum. Since the EPR spectrum of GdSL is the sum of subspectra of the individual EPR transitions, we attribute this field dependence to transition dependent phase relaxation. Using simulations of the EPR spectra and its decomposition into the individual transition subspectra, we isolated the phase relaxation of each transition and found that its rate increases with |ms|. We suggest that this mechanism is due to transient zero field splitting (tZFS), where its magnitude and correlation time are scaled down and distributed as compared with similar situations in liquids. This tZFS induced phase relaxation mechanism becomes dominant (or at least significant) when all other well-known phase relaxation mechanisms, such as spectral diffusion caused by nuclear spin diffusion, instantaneous and electron spin spectral diffusion, are significantly suppressed by matrix deuteration and low concentration, and when the temperature is sufficiently low to disable spin lattice interaction as a source of phase relaxation.

Optimization of pulsed DEER measurements for Gd-based labels: choice of operational frequencies, pulse durations and positions, and temperature.

In this work, the experimental conditions and parameters necessary to optimize the long-distance (≥ 60 Å) Double Electron-Electron Resonance (DEER) measurements of biomacromolecules labeled with Gd(III) tags are analyzed. The specific parameters discussed are the temperature, microwave band, the separation between the pumping and observation frequencies, pulse train repetition rate, pulse durations and pulse positioning in the electron paramagnetic resonance spectrum. It was found that: (i) in optimized DEER measurements, the observation pulses have to be applied at the maximum of the EPR spectrum; (ii) the optimal temperature range for Ka-band measurements is 14-17 K, while in W-band the optimal temperatures are between 6-9 K; (iii) W-band is preferable to Ka-band for DEER measurements. Recent achievements and the conditions necessary for short-distance measurements (<15 Å) are also briefly discussed.

Dielectric Resonator for Ka-Band Pulsed EPR Measurements at Cryogenic Temperatures: Probehead Construction and Applications.

The construction and performance of a Ka-band pulsed electron paramagnetic resonance (EPR) cryogenic probehead that incorporates dielectric resonator (DR) is presented. We demonstrate that the use of DR allows one to optimize pulsed double electron-electron resonance (DEER) measurements utilizing large resonator bandwidth and large amplitude of the microwave field B1 . In DEER measurements of Gd-based spin labels, use of this probe finally allows one to implement the potentials of Gd-based labels in distance measurements. Evidently, this DR is well suited to any applications requiring large B1-fields and resonator bandwidths, such as electron spin echo envelope modulation spectroscopy of nuclei having low magnetic moments and strong hyperfine interactions and double quantum coherence dipolar spectroscopy as was recently demonstrated in the application of a similar probe based on an loop-gap resonator and reported by Forrer et al. (J Magn Reson 190:280, 2008).

Pulsed dipolar spectroscopy distance measurements in biomacromolecules labeled with Gd(III) markers.

This work demonstrates the feasibility of using Gd(III) tags for long-range Double Electron Electron Resonance (DEER) distance measurements in biomacromolecules. Double-stranded 14- base pair Gd(III)-DNA conjugates were synthesized and investigated at K(a) band. For the longest Gd(III) tag the average distance and average deviation between Gd(III) ions determined from the DEER time domains was about 59±12Å. This result demonstrates that DEER measurements with Gd(III) tags can be routinely carried out for distances of at least 60Å, and analysis indicates that distance measurements up to 100Å are possible. Compared with commonly used nitroxide labels, Gd(III)-based labels will be most beneficial for the detection of distance variations in large biomacromolecules, with an emphasis on large scale changes in shape or distance. Tracking the folding/unfolding and domain interactions of proteins and the conformational changes in DNA are examples of such applications.

Distance measurements in model bis-Gd(III) complexes with flexible "bridge". Emulation of biological molecules having flexible structure with Gd(III) labels attached.

In this work, we continue to explore Gd(III) as a possible spin label for high-field Double Electron-Electron Resonance (DEER) based distance measurements in biological molecules with flexible geometry. For this purpose, a bis-Gd(III) complex with a flexible "bridge" was used as a model. The distances in the model were expected to be distributed in the range of 5-26 A, allowing us to probe the shortest limits of accessible distances which were found to be as small as 13 A. The upper distance limit for these labels was also evaluated and was found to be about 60 A. Various pulse duration setups can result in apparent differences in the distribution function derived from DEER kinetics due to short distance limit variations. The advantages, such as the ability to perform measurements at cryogenic temperatures and high repetition rates simultaneously, the use of very short pumping and observation pulses without mutual interference, the lack of orientational selectivity, as well as the shortcomings, such as the limited mw operational frequency range and intrinsically smaller amplitude of oscillation related to dipolar interaction as compared with nitroxide spin labels are discussed. Most probably the use of nitroxide and Gd-based labels for distance measurements will be complementary depending on the particulars of the problem and the availability of instrumentation.

HIGH-RESOLUTION EPR SPECTROSCOPY OF MO ENZYMES. SULFITE OXIDASES: STRUCTURAL AND FUNCTIONAL IMPLICATIONS.

Sulfite oxidases (SOs) are physiologically vital Mo-containing enzymes that occur in animals, plants, and bacteria and which catalyze the oxidation of sulfite to sulfate, the terminal reaction in the oxidative degradation of sulfur-containing compounds. X-ray structure determinations of SOs from several species show nearly identical coordination structures of the molybdenum active center, and a common catalytic mechanism has been proposed that involves the generation of a transient paramagnetic Mo(V) state through a series of coupled electron-proton transfer steps. This chapter describes the use of pulsed electron-nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopic techniques to obtain information about the structure of this Mo(V) species from the hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of nearby magnetic nuclei. Variable frequency instrumentation is essential to optimize the experimental conditions for measuring the couplings of different types of nuclei (e.g., (1)H, (2)H, (31)P, and (17)O). The theoretical background necessary for understanding the ESEEM and ENDOR spectra of the Mo(V) centers of SOs is outlined, and examples of the use of advanced pulsed EPR methods (RP-ESEEM, HYSCORE, integrated four-pulse ESEEM) for structure determination are presented. The analysis of variable-frequency pulsed EPR data from SOs is aided by parallel studies of model compounds that contain key functional groups or that are isotopically labeled and thus provide benchmark data for enzymes. Enormous progress has been made on the use of high-resolution variable-frequency pulsed EPR methods to investigate the structures and mechanisms of SOs during the past ~15 years, and the future is bright for the continued development and application of this technology to SOs, other molybdenum enzymes, and other problems in metallobiochemistry.

1H pulsed ENDOR and ESEEM evidence that the bis-imidazole complexes of iron(III) tetraphenylchlorin and tetraphenylporphyrin have the same order of g values, and the same electronic ground state.

The electronic structures of the bis-imidazole complexes of iron(III) tetraphenylporphyrin ([(TPP)Fe(ImH)(2)](+)) and iron(III) tetraphenylchlorin ([(TPC)Fe(ImH)(2)](+)) in frozen glassy solutions have been studied by the pulsed electron nuclear double resonance (ENDOR) technique of Mims and by electron spin-echo envelope modulation (ESEEM) spectroscopy. ESEEM spectra have been used to determine the orientation of the imidazole ligand planes with respect to the g tensor axes. In the ENDOR spectra, the manifestations of the implicit TRIPLE effect described and explained earlier by Doan et al. (J. Am. Chem. Soc. 1996, 118, 7014) were seen. In this work, the explicit expressions describing this effect were derived for the first time and used to successfully simulate the proton ENDOR spectra at the low- (LF) and high-field (HF) edges of the EPR spectrum. Using pulsed ENDOR, we have been able to determine the spin density distributions in the pi-systems of both tetrapyrroles and show that [(TPC)Fe(ImH)(2)](+) has the electronic orbital ground state (d(xy)())(2)(d(xz)(),d(yz)())(3), the same as that known for [(TPP)Fe(ImH)(2)](+), and the largest principal g value corresponds to the g tensor axis 3, which is normal to the heme plane. For the TPP complex, the g tensor axis 1, corresponding to the smallest principal g value, was found to be at an angle phi(1) of 30-35 degrees from the N-Fe-N axis, with the ligand planes rotated by the angle of 20-25 degrees in the opposite direction. For the TPC complex, phi(1) was found to be about 25 degrees from the direction N(I)-Fe-N(III), where N(I) corresponds to the nitrogen of the saturated pyrrole ring. The ligand planes in this complex were found to be oriented at an angle of about 10 degrees in the opposite direction.

Properties of the HYSCORE spin echo signal.

In hyperfine sublevel correlation spectroscopy (HYSCORE), the finite duration of the microwave pulses leads to an incomplete inversion of the electron spin magnetization by the third pulse, which results in a significant admixture of stimulated ESEEM to HYSCORE ESEEM. This virtually unavoidable contribution of stimulated ESEEM seriously hampers the analysis of the modulation amplitudes in HYSCORE. In this work, we analyze the properties of the spin echo signals contributing to the composite HYSCORE signal. Based on this analysis, we propose the strategies of HYSCORE data acquisition and processing that allow one to practically eliminate the contribution of the stimulated echo and make the HYSCORE ESEEM analyzable in quantitative terms.

ESEEM measurements with time-resolved detection of the entire ESE signal shape.

In this work we discuss the extension of electron spin-echo envelope modulation (ESEEM) measurements to the additional time dimension, the spin-echo coordinate. The time-resolved acquisition of the entire spin-echo signal shape retains information on the dependence of the ESEEM amplitude on the position within the ESE signal. Therefore, not only can such acquisition be completely substituted for the boxcar integration in ESEEM measurements, but it can also improve the performance of the ESEEM experiments in terms of obtaining correct modulation amplitudes and a better signal/noise ratio. Implementing such an acquisition in pulse-adjustable ESEEM measurements transforms these techniques into routine and convenient experiments used to increase the modulation amplitude.

ESEEM and Mössbauer studies of the ferriheme model compound bis(3-aminopyrazole)tetraphenylporphyrinatoiron(III) chloride, [TPPFe(NH2PzH)2]Cl.

A model heme complex, bis(3-aminopyrazole)tetraphenylporphinatoiron(III) chloride, [TPPFe (NH2PzH)2]Cl, for which the EPR g-values lead to a rhombicity V/delta = 1.2 if gzz is the largest g-value, have been investigated by electron spin echo envelope modulation (ESEEM) and Mössbauer spectroscopies. The ESEEM studies focus on the proton sum frequency peaks at near twice the proton Larmor frequency. Analysis of the distant proton peak (mainly due to the pyrrole-H) at exactly twice the proton Larmor frequency shows conclusively that gzz is aligned along the normal to the porphyrin plane, and thus the electron configuration is (dxy)2(dxz,dyz)3, with gzz > gyy > gxx. This system is thus another violation to Taylor's "proper axis system" rule. The near proton (the alpha-H and N-H of the axial ligands) peaks provide distance information for those protons from the metal. Magnetic Mössbauer studies of the same complex confirm the (dxy)2(dxz,dyz)3 ground state and indicate that, as is the case for cytochrome P450cam, Axx is the largest magnitude A-value, and is negative in sign. Other low-spin iron(III) porphyrinates also have Axx of negative sign, but usually the magnitude is only about half that of Azz, which is always positive in sign.