Velavan Kathirvelu

Impact of Electron-Electron Spin Interaction on Electron Spin Relaxation of Nitroxide Diradicals and Tetraradical in Glassy Solvents Between 10 and 300 K

To determine the impact of electron-electron spin-spin interactions on electron spin relaxation rates, 1/T1 and 1/Tm were measured for nitroxide monoradical, diradical, and tetraradical derivatives of 1,3-alternate calix[4]arenes, for two pegylated high-spin nitroxide diradicals, and for an azine-linked nitroxide diradical. The synthesis and characterization by SQUID (superconducting quantum interference device) magnetometry of one of the high-spin diradicals, in which nitroxides are conformationally constrained to be coplanar with the m-phenylene unit, is reported. The interspin distances ranged from about 5-9 A, and the magnitude of the exchange interaction ranged from >150 to >0.1 K. 1/T1 and 1/Tm were measured by long-pulse saturation recovery, three-pulse inversion recovery, and two-pulse echo decay at X-band (9.5 GHz) and Q-band (35 GHz). For a diradical with interspin distance about 9 A, relaxation rates were only slightly faster than for a monoradical with analogous structure. For interspin distances of about 5-6 A, relaxation rates in glassy solvents up to 300 K increased in the order monoradical < diradical < tetraradical. Modulation of electron-electron interaction enhanced relaxation via the direct, Raman, and local mode processes. The largest differences in 1/T1 were observed below 10 K, where the direct process dominates. For the three diradicals with comparable magnitude of dipolar interaction, 1/Tm and 1/T1 were faster for the molecules with more flexible structures. Relaxation rates were faster in the less rigid low-polarity sucrose octaacetate glass than in the more rigid 4:1 toluene/chloroform or in hydrogen-bonded glycerol glasses, which highlights the impact of motion on relaxation.

Impact of molecular size on electron spin relaxation rates of nitroxyl radicals in glassy solvents between 100 and 300 K

Electron spin lattice relaxation rates were measured for 12 nitroxyls with molecular weights between 144 and 438, and for galvinoxyl, 1,3-bisdiphenylene-2-phenylallyl (BDPA), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) in glassy sucrose octaacetate. Relaxation rates for polar nitroxyls also were measured in glassy sorbitol. Dependence on T where V eff is effective molecular volume and γ is a material-specific parameter, was used to compare processes. Values of V eff were determined based on molecular libration in glassy sucrose octaacetate (γ = 3.5), tumbling in viscous decalin at 233 K (γ = 4.7), or tumbling in heavy mineral oil (γ = 6.0). For nitroxyl relaxation there is a master curve: log(1/T 1) vs log( T) (γ = 0.89). The similarity of the values of γ for the Raman process and for the additional process that contributes at higher temperatures, and the absence of frequency dependence between X- and Q-band, support assignment of this additional process as a local mode. For these radicals the contributions from the local mode and the Raman process are correlated and follow trends in spin–orbit coupling. The temperature dependence of spin echo dephasing in sucrose octaacetate is dominated by rotation of methyl groups and by a motional process analogous to the Raman process.

A Spirocyclohexyl Nitroxide Amino Acid Spin Label for Pulsed EPR Spectroscopy Distance Measurements

Site-directed spin labeling and EPR spectroscopy offer accurate, sensitive tools for the characterization of structure and function of macromolecules and their assemblies. A new rigid spin label, spirocyclohexyl nitroxide alpha-amino acid and its N-(9-fluorenylmethoxycarbonyl) derivative, have been synthesized, which exhibit slow enough spin-echo dephasing to permit accurate distance measurements by pulsed EPR spectroscopy at temperatures up to 125 K in 1:1 water/glycerol and at higher temperatures in matrices with higher glass transition temperatures. Distance measurements in the liquid nitrogen temperature range are less expensive than those that require liquid helium, which will greatly facilitate applications of pulsed EPR spectroscopy to the study of structure and conformation of peptides and proteins.

Calix[4]arene nitroxide tetraradical and octaradical

1,3-Alternate calix[4]arene with para-phenylene spacers connecting nitroxide monoradicals and high-spin (S = 1) diradicals provides tetraradical and octaradical scaffolds that possess conformations with slow electron spin relaxation rates (1/T(1)). Such scaffolds may facilitate tuning of relaxation rates that are more favorable for MRI or DNP applications.

DEER distance measurement between a spin label and a native FAD semiquinone in electron transfer flavoprotein.

The human mitochondrial electron transfer flavoprotein (ETF) accepts electrons from at least 10 different flavoprotein dehydrogenases and transfers electrons to a single electron acceptor in the inner membrane. Paracoccus denitrificans ETF has the identical function, shares the same three-dimensional structure and functional domains, and exhibits the same conformational mobility. It has been proposed that the mobility of the alphaII domain permits the promiscuous behavior of ETF with respect to a variety of redox partners. Double electron-electron resonance (DEER) measurements between a spin label and an enzymatically reduced flavin adenine dinucleotide (FAD) cofactor in P. denitrificans ETF gave two distributions of distances: a major component centered at 4.2 +/- 0.1 nm and a minor component centered at 5.1 +/- 0.2 nm. Both components had widths of approximately 0.3 nm. A distance of 4.1 nm was calculated using the crystal structure of P. denitrificans ETF, which agrees with the major component obtained from the DEER measurement. The observation of a second distribution suggests that ETF, in the absence of substrate, adopts some conformations that are intermediate between the predominant free and substrate-bound states.

Rapid-scan EPR of immobilized nitroxides

X-band electron paramagnetic resonance spectra of immobilized nitroxides were obtained by rapid scan at 293 K. Scan widths were 155 G with 13.4 kHz scan frequency for (14)N-perdeuterated tempone and for T4 lysozyme doubly spin labeled with an iodoacetamide spirocyclohexyl nitroxide and 100 G with 20.9 kHz scan frequency for (15)N-perdeuterated tempone. These wide scans were made possible by modifications to our rapid-scan driver, scan coils made of Litz wire, and the placement of highly conducting aluminum plates on the poles of a Bruker 10″ magnet to reduce resistive losses in the magnet pole faces. For the same data acquisition time, the signal-to-noise for the rapid-scan absorption spectra was about an order of magnitude higher than for continuous wave first-derivative spectra recorded with modulation amplitudes that do not broaden the lineshapes.

A Signal-to-Noise Standard for Pulsed EPR

A 2 mm diameter by 10mm long cylinder of fused SiO2 (quartz) gamma-irradiated to 1 kGy with 60Co contains about 2x10(16) spins/cm3. It is proposed as a standard for monitoring signal-to-noise (S/N) performance of X-band pulsed EPR spectrometers. This sample yields S/N of about 25 on modern spin echo spectrometers, which permits measurement of both signal and noise under the same conditions with an 8-bit digitizer.

Electron transfer flavoprotein domain II orientation monitored using double electron-electron resonance between an enzymatically reduced, native FAD cofactor, and spin labels.

Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid β‐oxidation to the main respiratory chain. The crystal structure of human ETF bound to medium chain acyl‐CoA dehydrogenase indicates that the flavin adenine dinucleotide (FAD) domain (αII) is mobile, which permits more rapid electron transfer with donors and acceptors by providing closer access to the flavin and allows ETF to accept electrons from at least 10 different flavoprotein dehydrogenases. Sequence homology is high and low‐angle X‐ray scattering is identical for Paracoccus denitrificans (P. denitrificans) and human ETF. To characterize the orientations of the αII domain of P. denitrificans ETF, distances between enzymatically reduced FAD and spin labels in the three structural domains were measured by double electron‐electron resonance (DEER) at X‐ and Q‐bands. An FAD to spin label distance of 2.8 ± 0.15 nm for the label in the FAD‐containing αII domain (A210C) agreed with estimates from the crystal structure (3.0 nm), molecular dynamics simulations (2.7 nm), and rotamer library analysis (2.8 nm). Distances between the reduced FAD and labels in αI (A43C) were between 4.0 and 4.5 ± 0.35 nm and for βIII (A111C) the distance was 4.3 ± 0.15 nm. These values were intermediate between estimates from the crystal structure of P. denitrificans ETF and a homology model based on substrate‐bound human ETF. These distances suggest that the αII domain adopts orientations in solution that are intermediate between those which are observed in the crystal structures of free ETF (closed) and ETF bound to a dehydrogenase (open).

Electron spin relaxation rates for semiquinones between 25 and 295K in glass-forming solvents.

Electron spin lattice relaxation rates for five semiquinones (2,5-di-t-butyl-1,4-benzosemiquinone, 2,5-di-t-amyl-1,4-benzosemiquinone, 2,5-di-phenyl-1,4-benzosemiquinone, 2,6-di-t-butyl-1,4-benzosemiquinone, tetrahydroxy-1,4-benzosemiquione) were studied by long-pulse saturation recovery EPR in 1:4 glycerol:ethanol, 1:1 glycerol:ethanol, and triethanolamine between 25 and 295K. Although the dominant process changes with temperature, relaxation rates vary smoothly with temperature, even near the glass transition temperatures, and could be modeled as the sum of contributions that have the temperature dependence that is predicted for the direct, Raman, local mode and tumbling-dependent processes. At 85K, which is in a temperature range where the Raman process dominates, relaxation rates along the g(xx) (g approximately 2.006) and g(yy) (g approximately 2.005) axes are about 2.7-1.5 times faster than along the g(zz) axis (g=2.0023). In highly viscous triethanolamine, contributions from tumbling-dependent processes are negligible. At temperatures above 100K relaxation rates in triethanolamine are unchanged between X-band (9.5GHz) and Q-band (34GHz), so the process that dominates in this temperature interval was assigned as a local mode rather than a thermally activated process. Because the largest proton hyperfine couplings are only 2.2G, spin rotation makes a larger contribution than tumbling-dependent modulation of hyperfine anisotropy. Since g anisotropy is small, tumbling-dependent modulation of g anisotropy makes a smaller contribution than spin rotation at X-band. Although there was negligible impact of methyl rotation on T(1), rotation of t-butyl or t-amyl methyl groups enhances spin echo dephasing between 85 and 150K.