EARLY AND LATE M-INTERMEDIATE IN THE BACTERIORHODOPSIN PHOTOCYCLE - ASOLID-STATE NMR-STUDY

To enforce vectorial proton transport in bacteriorhodopsin (bR), it is necessary that there be a change in molecular structure between depro tonation and reprotonation of the chromophore-i.e., there must be at l east two different M intermediates in the functional photocycle. We pr esent here the first detection of multiple M intermediates in native w ild-type bacteriorhodopsin by solid-state NMR. Illumination of light-a dapted [zeta-N-15-Lys]-bR at low temperatures shifts the N-15 signal o f 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 illuminati on. The M state routinely prepared at the lower temperature, M-o, deca ys 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 uniq ue 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]r et 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 b ackbone of the protein, via [1-C-13]Val labeling, reveals a substantia l structural change between M-o and M-n including the relaxation of pe rturbations at some sites and the development of new perturbations at other sites. The combination of the change in the protein structure an d the increase in the pK(a) of the SE suggests that the demonstrated M -o-->M-n transition may function as the ''reprotonation switch'' requi red for vectorial proton transport.