Tyrosyl radicals are important in long-range electron transfer in several enzymes, but the protein environmental factors that control midpoint potential and electron transfer rate are not well understood. To develop a more detailed understanding of the effect of protein sequence, we have performed 14N and 15N electron spin echo envelope modulation (ESEEM) measurements on tyrosyl radical, generated either in polycrystalline tyrosinate or in its 15N-labeled isotopomer, by UV photolysis. 14N-ESEEM was also performed on tyrosyl radical generated in tyrosine-containing pentapeptide samples. Simulation of the 14N- and 15N-tyrosyl radical ESEEM measurements yielded no significant isotropic hyperfine splitting to the amine or amide nitrogen; the amplitude of the anisotropic, nitrogen hyperfine coupling (0.21 MHz) was consistent with a dipole-dipole distance of 3.0 A. Density functional theory was used to calculate the isotropic and anisotropic hyperfine couplings to the amino nitrogen in four different tyrosyl radical conformers. Comparison with the simulated data suggested that the lowest energy radical conformer, generated in tyrosine at pH 11, has a 76 degrees Calpha-Cbeta-C1'-C2' ring and a -73 degrees C-Calpha-Cbeta-C1' backbone dihedral angle. In addition, the magnitude, orientation, and asymmetry of the nuclear quadrupole coupling tensor were derived from analysis of the tyrosyl radical 14N-ESEEM. The simulations showed differences in the coupling and orientation of the nuclear quadrupole tensor, when the tyrosinate and pentapeptide samples were compared. These results suggest sequence- or conformation-induced changes in the ionic character of the NH bond in different tyrosine-containing peptides.