Identification of the Nitrogen Donor Hydrogen Bonded with the Semiquinone at the QH Site of the Cytochrome bo3 from Escherichia coli

The selective N-15 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 bo(3), ubiquinol oxidase. This nitrogen produces dominating contribution to X-Band N-14 ESEEM spectra. The 2D ESEEM (HYSCORE) experiments with the Q(H) site SQ in the series of selectively N-15 labeled bo(3) oxidase proteins have directly identified the N-epsilon of R71 as an H-bond donor. In addition, selective N-15 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.