On the two pathways of the M-intermediate formation in the photocycle of bacteriorhodopsin.

The flash-photolysis technique was used to study the photocycles of the wild-type bacteriorhodopsin (WT bR) and D96N mutant. Kinetics of the L-intermediate decay and M-intermediate formation at pH 7.0, 20 degrees C fit well a sum of two components having time constants, tau (1) = 60 microS and tau (2) = 250 microS, for the WT bR, and a sum of three components having time constants, tau (1) = 55 microS, tau (2) = 220 microS and tau (3) = 1 mS, for the D96N mutant. The fast component with a time constant of 1.4 microS was found in the photoresponse at 400 nm. It constituted 10% of the total amplitude and may be attributed to the K-->L transition. The component with tau = 1 mS was observed in the photocycle of the WT bR as a lag phase in the relaxation of the photoresponse at 400 nm. The difference absorbance minima, corresponding to the first (55-60 microS) and the second (220-260 microS) components of the M-formation, were located at 550 and 530 nm, respectively. The absorbance spectra, corresponding to the 1-mS-component of the M-formation of the D96N bR, may be represented as a superposition of spectra, corresponding to the first and the second components in the region of 460-700 nm. The effect of azide on the D96N bR revealed two azide-independent components in the decay of L-intermediate. Azide was shown to protonate all M-forms simultaneously. This indicates that the Schiff base pK rises almost immediately after deprotonation.(ABSTRACT TRUNCATED AT 250 WORDS)