SOLID-STATE C-13-NMR AND N-15-NMR INVESTIGATIONS OF THE N INTERMEDIATE OF BACTERIORHODOPSIN

Previous solid state C-13 NMR studies of bacteriorhodopsin (bR) have i nferred the C=N configuration and the protonation state of the retinal -lysine Schiff base (SB) linkage from the [13-C-13]retinal, [14-C-13]r etinal, and [epsilon-C-13]lysine-216 chemical shifts in the bR(555), b R(568), and M(412) states. Here we determine the C=N configuration and the protonation state of the N photointermediate that is cryotrapped along with the M photointermediate at high salt concentrations (0.1 M NaCl) and high pH (10.0). We obtained C-13 and N-15 SSNMR spectra of [ epsilon-N-15] lysine bR and [12-C-13]- and [13-C-13] retinal bR for sa mples illuminated under the above conditions. Two species are observed , both of which decay to bR(568) upon warming. One species has chemica l shifts identical to those obtained previously for M thermally trappe d in guanidine.HCl at high pH (Smith et al., 1989a; Farrar et al., 199 3). In the other species, the [epsilon(15)N]lysine and 13-C-13 chemica l shifts indicate that the SB is protonated, the 12-C-13 shift indicat es a 13=14 cis configuration, and the previously published [14-C-13]- and [epsilon(13)C]lysine shifts indicate a C=N anti configuration. The se results are consistent with other studies of the N photointermediat e.