SOLID-STATE NMR-STUDY OF [EPSILON-C-13]LYS-BACTERIORHODOSPIN - SCHIFF-BASE PHOTOISOMERIZATION

Previous solid state C-13-NMR studies of bacteriorhodopsin (bR) have i nferred the C = N configuration of the retinal-lysine Schiff base link age from the [14-C-13]retinal chemical shift (1-3). Here we verify the interpretation of the [14-C-13]-retinal data using the [epsilon-C-13] lysine 216 resonance. The epsilon-Lys-216 chemical shifts in bR555 (48 ppm) and bR568 (53 ppm) are consistent with a C=N isomerization from syn in bR555 to anti in bR568. The M photointermediate was trapped at pH 10.0 and low temperatures by illumination of samples containing eit her 0.5 M guanidine-HCl or 0.1 M NaCl. In both preparations, the [epsi lon-C-13]Lys-216 resonance of M is 6 ppm downfield from that of bR568. This shift is attributed to deprotonation of the Schiff base nitrogen and is consistent with the idea that the M intermediate contains a C= N anti chromophore. M is the only intermediate trapped in the presence of 0.5 M guanidine-HCl, whereas a second species, X, is trapped in th e presence of 0.1 M NaCl. The [epsilon-C-13]Lys-216 resonance of X is coincident with the signal for bR568, indicating that X is either C = N anti and protonated or C = N syn and deprotonated.