Interaction of the protonated Schiff base with the peptide backbone of valine 49 and the intervening water molecule in the N photointermediate of bacteriorhodopsin.

The effects of replacing Val49, Thr46, Asp96, and Phe219 in the cytoplasmic domain of bacteriorhodopsin on water O-H stretching vibrational bands and the amide I and imide II bands of the peptide backbone were examined in the M, N, and MN intermediates. This study is an extension of previous work on the L photointermediate [Yamazaki, Y., Tuzi, S., Saitô, H., Kandori, H., Needleman, R., Lanyi, J. K., and Maeda, A. (1996) Biochemistry 35, 4063-4068]. The O-H stretching bands at 3671 cm-1 in the M intermediate and at 3654 cm-1 in the N intermediate are shown to originate from the same water molecule. It is located in the region surrounded by the Schiff base, Val49, Thr46, and Phe219 in the M intermediate, and moves closer to Val49 in the M to N reaction. The peptide C-N bond between Val49 and Pro50 and the C=O bond of Val49 undergo perturbations upon formation of the N intermediate but not the M and N-like MN states in which the Schiff base is unprotonated. The carbonyl oxygen of Val49 is proposed to be the acceptor in H-bonding with the protonated Schiff base in the N intermediate. The results suggest that water molecules may be involved in this interaction in the cytoplasmic region, and may play a role in the accessibility change of the Schiff base in the L to M to N photocycle steps.