EPR and saturation transfer EPR spectra at high microwave field intensities

The eight unique EPR signals at the first and second harmonies of the Zeeman modulation are sensitive to the very slow rotational diffusion of spin labeled biomolecules when these signals are recorded under conditions of microwave saturation and finite Zeeman modulation frequencies and amplitudes. Such saturation transfer sensitive spectra have been employed to study contractile proteins, hemoproteins, enzymes, etc. When these species or their supramolecular complexes are characterized by correlation times in the range 10^?8 to 10^?3 s. Published computer simulation reproduce quite well spectra at the longer correlation times and the general sensitivity of spectra to changing rotational correlation time; however, agreement between experimental and calculated spectral shapes is poor for rotational correlation time on the order of 10^?7 s and the dependence of experimental spectra upon microwave field intensity is not reproduced. In the present communication we show that the previously reported discrepancy between experimental and calculated spectra is due to the neglect of higher order cou magnetic interactions modulated by the molecular motion and involving the spin-microwave field interaction. When these “pseudodiagonal” terms of the spin density equation are explicitly included, experimental spectral lineshapes, spectral line positions, and the ratios of amplitudes of the various signal components are quantitatively reproduced. Plots of the ratios of the heights of the high and low field spectral extrema suggest a procedure for calibrating microwave field intensities as these ratios are found experimentally and theoretically to be nearly a linear function of microwave field intensity for intensities in the range 0.15 to 0.5 G. The separation of low and high field extrema was observed to increase with increasing microwave field intensity, suggesting the need to carefully consider saturation effects when determining rotational correlation times from this separation.