Kinetic and spectroscopic evidence for an irreversible step between deprotonation and reprotonation of the Schiff base in the bacteriorhodopsin photocycle

The photocycles of wild-type bacteriorhodopsin and its D96N form were investigated with a gated multichannel analyzer. Reconstruction of the spectra of the photointermediates from the measured time-resolved difference spectra allowed evaluation of the kinetics; the data at pH 7 in the presence of 100 mM NaCl were best fitted by the scheme K in eqiulibrium L in equilibrium M1---M2 in equilibrium N in equilibrium O---BR plus N---BR [Váró, G., & Lanyi, J. K. (1990) Biochemistry 29, 2241-2250]. The proposed two M states and the M1---M2 reaction were necessitated by anomalies in the kinetics of the decay of K and L. Additional support was provided by a 4-nm blue-shift in the maximum of M in Triton X-100 solubilized bacteriorhodopsin during the photocycle; the kinetics of the shift were consistent with the time course of the proposed M1---M2 transition. In the D96N mutant, the M state is stabilized, and the resulting equilibrium mixture for the intermediates could be evaluated with greater precision. The concentration ratio of L to M at the equilibrium was estimated to be no higher than 0.01. This requires the ratio of forward/reverse rates for the M1 to M2 conversion to be at least 200, i.e., a virtually irreversible reaction. Consistent with an earlier report, the data at lower pH and in the absence of NaCl are different and suggest the existence of a second L species; we propose that it is in equilibrium with M2.