A Role for Internal Water Molecules in Proton Affinity Changes in the Schiff Base and Asp85 for One-way Proton Transfer in Bacteriorhodopsin[dagger]

Photochemistry and Photobiology, Jul/Aug 2008 by Morgan, Joel E, Gennis, Robert B, Maeda, Akio

L is the state immediately before the first proton transfer. The L-minus-BR spectrum can be recorded at 170 K as a steady state under continuous illumination. The spectrum exhibits a broad feature between 3550 and 3450 cm^sup -1^, which is composed of at least three water O-H vibration bands (17,18). In this spectrum, the positive bands (due to L) are more intense than the negative bands (BR) reflecting the fact that water molecules become polarized upon L formation. No vibrational hands of this nature appear in either the K intermediate at 80 K or K^sub L^ at 135 K (19). The intensity of these bands is affected by mutants of residues located on the cytoplasmic side of the Schiff base: Thr46, Asp96, Leu93 and Va149).

The details of these mutant studies have been reviewed previously (20,21), but will be repeated concisely below to emphasize the effect of the waler cluster on the protonated state of the Schiff base. The intensity of the water bands in the L-minus-BR spectrum becomes smaller in T46V, but is restored in T46V/D96N in parallel with a shift of the L-to-M equilibrium to M, which is also reversed in the double mutant (22). These mutations did not affect the vibrational bands of water at 3643 cm^sup -1^ or of Asp85 at 1761 cm^sup -1^, both of which are on the extracellular side of the Schiff base. The shift of equilibrium toward L was also observed in V49A and L93M, mutants in which there is an increase in intensity of the water vibrational bands (18,23) apparently related to removal of steric hindrance, which then allows accommodation of the water cluster. This effect of the side chain of Leu93 has also been suggested by molecular dynamics calculations (24) and crystallographic analysis (25). A water cluster forms in the cavity surrounded by the protein stretching from the Schiff base to the region around Asp96 and Gly220 (26). This structure has sufficient structural integrity to be almost completely preserved at 80 K, even without the direct support of the Schiff base. This can be observed in the L' state, in which the Schiff base N-H is oriented toward Asp85, probably through Water402. However, this structure cannot be maintained if the sample is warmed to 170 K, indicating that the hydrogen bonding interaction of the Schiff base N-H that orients toward the cytoplasmic side is of primary importance for the L structure, including the water cluster (27). These results suggest that the water cluster, together with the surrounding protein environment on the cytoplasmic side, works to regulate the L�M equilibrium under the influence of the protonated Schiff base N-H.

The structure proposed on the basis of our FTIR results can be depicted on the basis of the X-ray structural model in protein databank entry 1ucq (25), which contains two water molecules with high occupancy and a space to accommodate two additional water molecules exlending from the Schiff base 10 the region around Thr46 on the cytoplasmic side. However, the FTIR data are difficult to reconcile with other models: 1r3p (28) and 2ntw (29), which do not show such a water cluster. Also, the Schiff base N-H in 2ntw (29) is connected to Asp85 through Watcr402 on the extracellular side. This is hard to reconcile with our proposed structure of L.