Pulsed ELDOR

M. Bennati, A. Weber, J. Antonie, D.L. Perlstein, J. Robblee and J. Stubbe, Pulsed ELDOR spectroscopy measures the distance between the two tyrosyl radicals in the R2 subunit of the E. coli ribonucleotide reductase, J. Am. Chem. Soc., 2003, 125, 14988. 

H. Mino and T. Ono, Applications of pulsed ELDOR-detected NMR measurements to studies of photosystem II Magnetic characterization of Yd tyrosine radical and Mn2+ bound to the high-affinity site, Appl. Magn. Reson., 2003, 23, 571. 

The two centres are connected by a flexible linkage that does not have well defined secondary structure. Pulsed ELDOR experiments were performed at 4.2 K using a one-loop-one-gap resonator. The narrow Mo(V) signal was pulsed at... 

Kay, C. W. M., Mkami, H. E., Cammack, R., and Evans, R. W. (2007). Pulsed ELDOR determination of the intramolecular distance between the metal binding sites in dicupric human serum transferrin and lactoferrin. J. Am. Chem. Soc. 129, 4868. 

Several pulse methods were developed for estimation distances between two slowly-relaxing spins. In a pulse electron-electron double resonance (PELDOR) technique a spin echo is created by a two-pulse sequence at one microwave frequency. The timing of a pulse at a second microwave frequency is varied (Milov et al., 1998). This method is suitable for analysis of weak dipolar interactions. 3-pulse PELDOR with all three pulses at the same microwave frequency ( 2 + 1 sequence) was proposed by Raitsimling and his co-workers (2000). A specific feature of the 2 + 1 technique is suppression of dipolar interaction of randomly distributed spins, which allows the selection of a dipolar interaction between radicals. Using a 4- pulse experiments it was possible to eliminate an inherent dead experimental deadtime that limits the magnitude of the dipolar interaction in 2 + 1 sequence and in 3-pulse ELDOR experiments (Pannier et al., 2000). 

Jeschke G, Panek G, Godt A, Bender A, Paulsen H (2004) Data analysis procedures for pulse ELDOR measurements of broad distance distributions. Appl Magn Reson 26 223-244... 

Jeschke G, Schlick S (2006) Spatial distribution of stabilizer-derived nitroxide radicals during thermal degradation of poly(acrylonitrile-butadiene-styrene) copolymers a unified picture from pulsed ELDOR and ESR imaging. Phys Chem Chem Phys 8 4095 103... 

CW spectrometers are summarised in a book by Kevan and Kispert [4], The technique has been revived in pulsed ELDOR experiments to obtain distances between specific spin-labelled sites in polymers and biopolymers [5, 6],... 

Electron-electron Pulsed ELDOR 15-100 dipolar coupling Electron-electron Point dipole... 

A commonly employed method is by pulsed ELDOR, see [5] for a review. The modulation frequency measured from the ELIX)R spectrum equals the value of D ... 

Pulsed ELDOR. Distances between electron spins can be measured by double electron-electron resonance (DEER) experiments such as the four-pulse experiment illustrated in Figure 6 (34). Similar to measurements of electron-nucleus distances, this technique is based on the r dependence of the magnetic dipole interaction between electron spins and can determine larger distances, in the range 1.5-5 nm. One of two spins (color-coded green, observer) is observed by a refocused primary echo with fixed interpulse delays ti and t2, so that relaxation does not induce variations in the echo amplitude during the experiment. The second spin (color-coded red, pumped) imposes a local dipole field at the site of the first spin, with a magnitude that depends on the distance. At a variable delay t with respect... 

Fig. 3. Pulsed ELDOR (DEER) sequences, (a) Basic PELDOR experiment. Inteipulse delay T is fixed, inteipulse delay t is varied, and echo intensity is recorded, (b) Four-pulse DEER. In the constant-time version, interpulse delays X and X2 are fixed, interpulse delay f is varied, and the intensity of the second echo (sohd line) is recorded. For the variahle-time version, see the text.

With the basic pulsed ELDOR (PELDOR) sequence shown in Fig. 3a, such a measurement at t = 0 corresponds to overlapping pump and observer pulses. This leads to signal distortions unless the two frequencies are applied to two well-isolated modes of a bimodal resonator. Furthermore, pulses at the two frequencies have to be amplified in two separate high-power amplifiers. The requirement for such specialized hardware and the restriction to a fixed frequency difference imposed by the bimodal resonator is overcome by using the four-pulse double electron-electron resonance (DEER) experiment (Fig. 4b). 

Fig. 4. Energy level scheme, transition frequencies, and coherence transfer in pulsed ELDOR experiments, (a) Sitnation before the pump pulse, (b) Situation after the pump pulse.

Scattering and pulsed ELDOR differ profoundly in formation of the signal. Scattered waves totally cancel by destructive interference if there are no regularities in the strncture. For particles whose positions are uncorrelated (particles homogeneonsly distributed in a matrix) the structure factor is a constant, S(q) = 1. In contrast, homogeneously distributed spins in pulsed ELDOR contribute a background fnnction B(t),... 

The primary result of a pulsed ELDOR measurement is a distribution of dipolar cou-phngs d. This information is contained either in the primary time-domain data (variation of echo intensity as a function of dipolar evolution time t) or in the dipolar spectrum obtained from these data by Fourier transformation. Time- and frequency-domain data contain exactly the same information, since Fourier transformation is a linear operation. However, some features are easier to recognize in time domain (e.g., quahty of least-squares fitting of the data) and others in frequency domain (e.g., orientation selection by missing parts of the Pake pattern). 

Fig. 15. Pulsed ELDOR (DEER) distance measurements on the ionic spin-probe TEMPO-4-carboxylate attached to ionic clusters in ionically modified diblock copolymers, (a) Schematic structure of a monoionic polystyiene-polyisoprene diblock copolymer modified by sulfonate end groups on the polyisoprene bloek (sample series S). (b) Schematic structure of an a,0)-zwitterionic polystyrene- ly-isoprene diblock copolymer modified by a quaternary ammonium end group on the polystyrene block and a sulfonate end group on the polyisoprene block (sample series Z). (c) Schematic structures of the polymer chains. Tlie solid line corresponds to the harder block polystyrene, the dotted line to the softer block polyisoprene. (d) Dependence of ionic cluster size (ri) and intercluster distance (r2> on molecular weight. Squares correspond to sample series Z, circles to sample series S, and diamonds to monionic homopolymers (polystyrene modified with quaternary ammonium end groups). The dotted and dashed lines are fits of a constant function. The solid line is the best-fit scaling law r2 = 2.09...

Figure 21. Pulsed ELDOR data of E. coli R2 and Rnr2Rnr4 from yeast (A) low-temperature (5 K) spin-echo detected spectrum of Tyr in E. coli R2, (B) time traces normalized with the echo signal intensity at zero time, and (C) frequency spectrum. Reprinted with permission from [334]. Copyright 2003, American Chemical Society.

The size of the spin-spin interaction of the [NiFe] center and the [3Fe4S] cluster (S = Vi) is within the range that can be studied by pulse ELDOR spectroscopy. This PELDOR technique allows measurement of the spin-spin interaction and a determination of the effective distance between the two electron spins. Measurements have so far only been performed for D. vulgaris Miyazaki F hydrogenase [94] on the as-isolated enzyme (30% Ni-A and 70% Ni-B). The spin delocalization over the [3Fe4S] cluster had to be included for correct data analysis. Spin projection coefficients have been determined that indicate that the largest amount of eleetron spin density is located on the iron closest to the [NiFe] center. 

A unified picture from pulsed ELDOR and ESRI has been obtained recently. ... 

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