The high-affinity Q(H) ubiquinone-binding site in the bo(3) ubiquinol oxidase from Escherichia coli has been characterized by an investigation of the native ubiquinone radical anion Q(H)(.-) 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(H)(.-) with a nitrogen nucleus from the surrounding protein matrix. From analysis of the experimental data, the N-14 nuclear quadrupolar parameters have been determined: kappa = e(2)qQ/4h = 0.93 MHz and eta = 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 tu a protein backbone nitrogen. This result is discussed with regard to the location and potential function of Q(H) in the enzyme.