No abstract

Four doubly spin-labeled variants of human carbonic anhydrase II and corresponding singly labeled variants were prepared by site-directed spin labeling. The distances between the spin labels were obtained from continuous-wave electron paramagnetic resonance spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of line-shape broadening, and computer simulation of line-shape changes. Distances also were determined by four-pulse double electron-electron resonance. For each variant, at least two methods were applicable and reasonable agreement between methods was obtained. Distances ranged from 7 to 24 A. The doubly spin-labeled samples contained some singly labeled protein due to incomplete labeling. The sensitivity of each of the distance determination methods to the non-interacting component was compared.

Electron spin lattice relaxation rates (1/T1 ) were measured as a function of temperature at two or three microwave frequencies for three S = 1/2 species in temperature ranges with different dominant relaxation processes. Between 10 and 50 K the contribution from the direct process to the relaxation rate was substantially greater at 94 than at 9.5 GHz for a vanadyl porphyrin doped into zinc tetratolylporphyrin. For bis(diethyldithiocarbamato)copper(TI) doped into the diamagnetic Ni(II) analog the relaxation rate between 25 and 100 K is dominated by the Raman process and exhibits little frequency dependence between 9.2 and 94 GHz. For 4-hydroxy-2,2,6,6-tetramethylpiperidinoloxy (tempol) doped into a diamagnetic host the relaxation rate between about 40 and 100 K is dominated by the Raman process. In this temperature range, relaxation rates at 3.2, 9.2, and 94 GHz exhibit little frequency dependence. Above about 130 K, the relaxation rate for tempol decreases in the order S-band > X-band > W-band. The relaxation rates in this temperature range fit a model in which ]IT, is dominated by a thermally activated process that is assigned as rotation of the methyl groups on the nitroxyl ring.

No abstract

Radicals genemted by y-irradiation of malonic acid and methyl malonic acid were studied asa function of temperature by inversion recovery, echo-detected saturation recovery and electronelectron double resonance (ELDOR) at X-band, and by continuous-wave saturation recovery at Xband and S-band. ELDOR reductions for malonic acid radical in polycrystalline and single-crystal samples indicate that nuclear spin relaxation is faster than both electron spin relaxation and cross relaxation between 93 and 233 K. Deuteration of the carboxylate protons caused the maximum ELDOR reduction to shift from about 110 to 150 K, consistent with the assignment of the rapid nuclear spin relaxation to hydrogen-bonded proton dynamies. ELDOR enhancements for both radicals formed in methyl malonic acid indicate that cross relaxation is faster than both electron spin relaxation and nuclear spin relaxation between 77 and 220 K. Enhanced cross relaxation and electron spin relaxation ate attributed to the rotation of methyl groups at a rate comparable to the electron Larmor frequency. The temperature dependence of the enhancement of 1/T~, was analyzed to determine the activation energies for methyl rotation. The same radical is formed in irradiated methyl malonic acid and L-alanine, but the barrier to rotation of the ct-methyl is 500 K in the methyl malonic acid host and 1500 K in the L-alanine host, which indicates a large impact of the lattice on the barrier to rotation.