ON THE 2 FORMS OF BACTERIORHODOPSIN

In our previous work [(1993) FEBS Lett. 313, 248-250; (1993) Biochem. Int. 30, 461-469] M-intermediate formation of wild-type bacteriorhodop sin was shown to involve two components differing in time constants (t au(1)=60-70 mu s and tau(2)=220-250 mu s), which were suggested to ref lect two independent pathways of M-intermediate formation. The contrib ution of the fast M was 4-times higher than the slow one. Our present research on M-intermediate formation in the D115N bacteriorhodopsin mu tant revealed the same components but at a contribution ratio of 1:1. Upon lowering the pH, the slow phase of M-formation vanished at a pK o f 6.2, and in the pH region 3.0-5.5 only the M-intermediate with a ris e time of 60 mu s was present. A 5-6 h incubation of D115N bacteriorho dopsin at pH 10.6 resulted in the irreversible transformation of 50% o f 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 bacteriorhodopsin contained 100% fast M-intermediate. The disappearance of the 250-mu s phase concomit ant with bR460 formation indicates that at neutral pH bacteriorhodopsi n exists as two spectroscopically indistinguishable forms.