Early and late M intermediates in the bacteriorhodopsin photocycle: A solid-state NMR study

To enforce vectorial proton transport in bacteriorhodopsin (bR), it is necessary that there be a change in molecular structure between deprotonation and reprotonation of the chromophore-i.e., there must be at least two different M intermediates in the functional photocycle. We present here the first detection of multiple M intermediates in native wild-type bacteriorhodopsin by solid-state NMR. Illumination of light-adapted [zeta-N-15-Lys]-bR at low temperatures shifts the N-15 signal of the retinal Schiff base (SB) downfield by about 150 ppm, indicating a deprotonated chromophore. In 0.3 M Gdn-HCl at pH 10.0, two different M states are obtained, depending on the temperature during illumination. The M state routinely prepared at the lower temperature, M-o, decays to the newly observed M state, M-n, and the N intermediate, as the temperature is increased. Both relax to bR(568) at 0 degrees C. A unique reaction sequence is derived: bR(568)-->M-o-->(M-n+N)-->bR(568). M-o and M-n have similar chemical shifts at [12-C-13]ret, [14-C-13]ret, and [epsilon-C-13]Lys216, indicating that M-n, like M-o, has a 13-cis and C=N anti chromophore. However, a small splitting in the [14-C-13]ret signal of M-o reveals that it has at least two substates. The 7 ppm greater shielding of the SE nitrogen in M-n compared to M-o suggests an increase in basicity and/or hydrogen bonding. Probing the peptide backbone of the protein, via [1-C-13]Val labeling, reveals a substantial structural change between M-o and M-n including the relaxation of perturbations at some sites and the development of new perturbations at other sites. The combination of the change in the protein structure and the increase in the pK(a) of the SE suggests that the demonstrated M-o-->M-n transition may function as the "reprotonation switch" required for vectorial proton transport.