The Temperature Dependence of Nitroxide Spin–Label Interaction Parameters: a High-Field EPR Study of Intramolecular Motional Contributions

Abstract

High-field W-band electron paramagnetic resonance (EPR) spectroscopy was utilized to study the temperature dependence of the magnetic interaction parameters (g-, hyperfine-, quadrupole tensors) of two types of doublet-state nitroxide spin probes in glass-forming ortho-terphenyl solution: a five-membered ring system of pyrroline type (model for the commonly used methane thiosulfonate spin label) and a six-membered ring system of piperidine type (model for the commonly used TOAC spin label). The analysis of the g-and hyperfine tensors in terms of their isotropic and anisotropic parts reveals at least two mechanisms of motion that are responsible for the temperature dependence of the interaction parameters. The first mechanism is attributed to the overall small-angle motion of the nitroxide molecule in the glassy matrix; it leads to an averaging of the anisotropies of the EPR parameters. The second mechanism originates in an intramolecular out-of-plane motion of oxygen in the nitroxide group. This type of motion is evidenced by comparing the experimental findings for the spin-interaction parameters with the results of density functional theory calculations. The harmonic oxygen out-of-plane vibrations result in a variation of both the isotropic and anisotropic parts of the g-and hyperfine tensors. In contrast, the quadrupole tensor is not influenced by this vibration mechanism in the temperature range under study (90-240 K). Consequences of the applicability of such typical nitroxide radicals for probing details of their protein environment and for studying librational dynamics in frozen solutions are discussed.

Multifrequency Pulsed EPR and the Characterization of Molecular Dynamics

“…The large differences in rates of interconversion of ring conformations for 5- and 6-member ring nitroxides (Rockenbauer, Korecz, & Hideg, 1993) and estimates of lifetimes substantially longer than 10 −10 s at 298 K (Barbon, Brustolon, Maniero, Romanelli, & Brunel, 1999) indicate that interconversion of ring conformations is too slow to be assigned as the thermally-activated process for nitroxide relaxation. Recent molecular dynamics calculations have shown that the intramolecular out-of-plane motion of the N-O moiety is a ‘soft’ mode (Pavone, Biczysko, Rega, & Barone, 2010; Savitsky, Plato, & Mobius, 2010). For both 5- and 6-member ring nitroxides the angle between the N-O group and the plane of the molecule can vary by ±10° within a low-energy range of about 0.5 kcal/mole (Savitsky et al, 2010).…”

“…Recent molecular dynamics calculations have shown that the intramolecular out-of-plane motion of the N-O moiety is a ‘soft’ mode (Pavone, Biczysko, Rega, & Barone, 2010; Savitsky, Plato, & Mobius, 2010). For both 5- and 6-member ring nitroxides the angle between the N-O group and the plane of the molecule can vary by ±10° within a low-energy range of about 0.5 kcal/mole (Savitsky et al, 2010). This angular variation is sufficient to cause substantial changes in the isotropic g and A values.…”

Fast motion in molecular solids at low temperatures: Evidence from a pulsed electron paramagnetic resonance study of nitroxyl radical relaxation

“…The experimental evidence derived from these analyses seems to overrule the possibility of methyl group dynamics being relevant in the shortening of T M below 20 K. 41 This reasoning is supported by data from the literature showing that for a similar nitroxyl radical embedded in a glassy matrix, the parameters of hyperfine interaction measured at 0.4T g were identical to those measured at 0.7T g . 30 In addition, the importance of intramolecular motion such as the out-of-plane vibration of the radical molecule itself as a source of electron spin relaxation 30 is not a factor within the context of this study because the energies involved are smaller than 20 K (1.7 meV) and additional ENDOR lines did not appear down to 5 K (0.05T g ). 17 In order to attempt to explain the experimental results, we sought a physical mechanism capable of explaining the increase in 1/T M below 20 K while still being consistent with the transition from slow to fast dynamics with respect to the time scale of the EPR experiment.…”

“…14,30 Glassy and crystalline states of ethanol were prepared as reported 31 following the procedure used in specific-heat measurements. 18 In brief, the ethanol glass was prepared from liquid ethanol, kept at room temperature, by rapid (∼10 s) insertion of the sample into the EPR cryostat, precooled to 80 K. The formed ethanol glass was left for equilibration for at least 30 min, before the EPR measurements were started.…”

“…[27][28][29] The accuracy of this procedure increases with increasing EPR detection frequency (W-band and higher frequencies) due to the improved separation of the tensor elements at canonical orientations in the magnetic field. 28,30 In this approach, the Hamiltonian, H , for electron spin S exhibiting hyperfine interaction with nuclear spin I in an external magnetic field, B, is approximated in the EPR principal axes (x, y, z) coordinate system by…”