The two consecutive M substates in the photocycle of bacteriorhodopsin are affected specifically by the D85N and D96N residue replacements

The photocycle of the proton pump bacteriorhodopsin contains two consecutive intermediates in which the retinal Schiff base is unprotonated; the reaction between these states, termed M1 and M2, was suggested to be the switch in the proton transport which reorients the Schiff base from D85 on the extracellular side to D96 on the cytoplasmic side (Varo and Lanyi, Biochemistry 30, 5016-5022, 1991). At pH 10 the absorption maxima of both M1 and M2 could be determined in the recombinant D96N protein. We find that M1 absorbs at 411 nm as do M1 and M2 in wild-type bacteriorhodopsin, but M2 absorbs at 404 nm. Thus, in M2 but not M1 the unprotonated Schiff base is affected by the D96N residue replacement. The connectivity of the Schiff base to D96 in the detected M2 state, but not in M1, is thereby established. On the other hand, the photostationary state which develops during illumination of D85N bacteriorhodopsin contains an M state corresponding to M1 with an absorption maximum shifted to 400 nm, suggesting that this species in turn is affected by D85. These results are consistent with the suggestion that M1 and M2 are pre-switch and post-switch states, respectively.