Water rearrangement around the Schiff base in the late K (K_L) intermediate of the bacteriorhodopsin photocycle

Previous FTIR studies at room temperature have distinguished K_L (late K) from K (early K) as distinct photocycle intermediates of all-trans bacteriorhodopsin differentiated by the prominence of their hydrogen out-of-plane bending vibration (HOOP) bands at 985 and 957 cm^-1, respectively. Earlier we showed that in the next photointermediate, L, the C₁₅,N-HOOP appears as a set of several bands at around 1064 cm^-1, which were shown to be due to the interaction of the Schiff base with water (Maeda et al. Biochemistry 2002, 41, 3803). The present study examines water rearrangement around the Schiff base in K and K_L by analysis of the HOOP bands and the water vibration bands. (1) Studies of the thermal transformation of the primary photoproduct K formed at 80 K upon warming to 135 and 170 K confirm that a K-like photoproduct formed at 135 K is an intermediate between K and L. From its HOOP band at 985 cm^-1, this photoproduct can be assigned to K_L. (2) In K_L, as in L, the C₁₅,N-HOOP band is affected upon replacement of H₂O by H₂¹⁸O water indicating the Schiff base's interaction with water. This feature was not observed for K. (3) The water molecule that is affected by the T46V mutation undergoes a perturbation in K_L as in L, and to a smaller extent in K. However, in K_L, the water does not exhibit the intense band seen in L. (4) A water molecule near Asp85 (most likely Water401), which is perturbed only slightly in K, is more strongly perturbed in K_L and in L.