Orientation dependence of electric field effects on the g factor of nitroxides measured by 220 GHz EPR

The spectroscopic properties of stable nitroxide radicals, including the ma gnetic properties of the impaired electron, are known to be sensitive to lo cal electric fields. The most accessible parameter has been the isotropic N -14 hyperfine splitting a(N), which is readily measured using conventional electron paramagnetic resonance (EPR) spectroscopy. High-frequency, high-fi eld EPR may be used to gain additional information about the electrostatic microenvironment of a nitroxide by revealing subtle influences on the nitro xide g tensor. In the present work, two nitroxide radicals with a local ele ctric field E-loc provided by internal ionizable groups are studied as a fu nction of pH by 220 GHz EPR. The three principal g values are resolved by i mmobilizing the probes in pH-adjusted poly(vinyl alcohol) films. Both nitro xides exhibit observable shifts in g(x) and g(y) as a function of pH, from which the g shifts may be calibrated with respect to E-loc The calibration shows that nitroxide probes are sensitive to field magnitudes that are typi cally found in proteins, and may provide a useful means to measure such fie lds quantitatively. In addition, differences between the two probes in the relative effect of E-loc upon g(y) are observed, which suggests that that t he g tensor may be sensitive to the orientation of the local field as well as its magnitude. Finally, the measured sensitivity to internal electric fi elds implies that high-field EPR of nitroxides should also be sensitive to externally applied electric field, i.e., should exhibit a linear electric f ield effect (LEFE). Although the LEFE at conventional EPR Fields is well ch aracterized for metal centers, our results suggest that high-field EPR shou ld enable its observation in organic radicals as well.