The high-affinity Q ubiquinone-binding site in the bo ubiquinol oxidase from Escherichia coli has been characterized by an investigation of the native ubiquinone radical anion Q by pulsed electron paramagnetic resonance (EPR) spectroscopy. One- and two-dimensional electron spin-echo envelope modulation (ESEEM) spectra reveal strong interactions of the unpaired electron of Q with a nitrogen nucleus from the surrounding protein matrix. From analysis of the experimental data, the N nuclear quadrupolar parameters have been determined: κ = eqQ/4h = 0.93 MHz and η = 0.50. This assignment is confirmed by hyperfine sublevel correlation (HYSCORE) spectroscopy. On the basis of a comparison of these data with those obtained previously for other membrane-protein bound semiquinone radicals and model systems, this nucleus is assigned to a protein backbone nitrogen. This result is discussed with regard to the location and potential function of Q in the enzyme.