In our previous work [(1993) FEBS Lett. 313,248-250; Biochem. Int. 30,[461][462][463][464][465][466][467][468][469] M-intermediate formation of wild-type bacteriorhodopsin was shown to involve two components differing in time constants (z, = 6&7Ops and z2 = 22&25Ops), which were suggested to reflect two independent pathways of M-intermediate formation. The contribution of the fast M was 4-times higher than the slow one. Our present research on M-intermediate formation in the Dll5N bacteriorhodopsin mutant revealed the same components but at a contribution ratio of 1:l. Upon lowering the pH, the slow phase of M-formation vanished at a pK of 6.2, and in the pH region 3.G5.5 only the M-intermediate with a rise time of 60 pus was present. A 5-6 h incubation of D115N bacteriorhodopsin at pH 10.6 resulted in the irreversible transformation of 50% of the protein into a form with a difference absorbance maximum at 460 nm. This form was stable at pH 7.5 and had no photocycle, including M-intermediate formation. The remaining bactenorhodopsin contained 100% fast M-intermediate. The disappearance of the 250;~s phase concomitant with bR460 formation indicates that at neutral pH bacteriorhodopsin exists as two spectroscopically indistinguishable forms.