Identification of the nitrogen donor hydrogen bonded with the semiquinone at the Q_H site of the cytochrome bo₃ from Escherichia coli

The selective ¹⁵N isotope labeling was used for the identification of the nitrogen involved in a hydrogen bond formation with the semiquinone in the high-affinity Q_H site in the cytochrome bo₃ ubiquinol oxidase. This nitrogen produces dominating contribution to X-Band ¹⁴N ESEEM spectra. The 2D ESEEM (HYSCORE) experiments with the Q_H site SQ in the series of selectively ¹⁵N labeled bo₃ oxidase proteins have directly identified the N_ε of R71 as an H-bond donor. In addition, selective ¹⁵N labeling has allowed us for the first time to determine weak hyperfine couplings with the side-chain nitrogens from all residues around the SQ. Those are reflecting a distribution of the unpaired spin density over the protein in the SQ state of the quinone processing site.