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

ABSTRACT

Light-induced proton pumping in bacteriorhodospin is carried out through five proton transfer steps. We propose that the proton transfer to Asp85 from the Schiff base in the L-to-M transition is accompanied by the relocation of a water cluster on the cytoplasmic side of the Schiff base from a site close to the Schiff base in L to the Phe219-Thr46 region in M. The water cluster present in L, formed at 170 K, is more rigid than that at room temperature. This may be responsible for blocking the conversion of L to M at 170 K. In the photocycle at room temperature, this water cluster returns to the site close to the Schiff base in N, with a rigid structure similar to that of L at 170 K. The increase in the proton affinity of Asp85, which is a prerequisite for the one-way proton transfer in the M-to-N transition, is suggested to be facilitated by a structural change which disrupts interactions between Asp212 and the Schiff base, and between Asp212 and Arg82. We propose that this liberation of Asp212 is accompanied by a rearrangement of the structure of water molecules between Asp85 and Asp212, stabilizing the protonated Asp85 in M.

INTRODUCTION

Bacteriorhodopsin is an excellent enzyme system in which to investigate the underlying chemistry of protein function. The reaction can he started by light, which is a noninvasive trigger of the protein function, and allows precise comparison of the intermediates to the initial unphotolyzed state having the all-trans retinal chromophore (BR). Fourier transform infrared spectroscopy (FTIR) studies are especially powerful for investigating changes in the polar groups around active sites when combined with structural information obtained by X-ray diffraction studies.

Light causes the trans-to-cis isomerization of the C^sub 13^ = C^sub 14^ bond of the retinal chromophore, leading to a sequence of photointermediates that unfold over the course of femtoseconds to milliseconds, resulting in conservation of a fraction of the photon's energy by means of the electrogenic transfer of a proton across the bacterial membrane. Unidirectional proton pumping by bacteriorhodopsin is the result of the sum of intramolecular proton transfer processes, each of which is closely synchronized with the transitions between the photointermediates, L, M, N and O (concisely summarized with references in Morgan et al. [1]). Residues and water molecules relevant to the current studies are shown in Fig. 1 on the basis of the crystallographic structure of protein databank entry 1c3w (2). The first proton transfer from the Schiff base to Asp85, on the extracellular side of the Schiff base, occurs in the L-to-M transition. Concomitant with this, a proton is released (3) from the proton release group (PRG), which is composed of Glu204, Glu194, Arg82, Tyr57 and water molecules surrounded by these residues (4). The deprotonated Schiff base of M then accepts a proton from Asp96, on the opposite side of the Schiff base, resulting in the formation of N. A proton is taken up by Asp96 from the cytoplasm in the N-to-O transition, and this is also accompanied by the cis-to-trans reisomerization of the C^sub 13^ = C^sub 14^ bond. The final step is the proton transfer from Asp85 to the PRG, resetting the structure to BR. In the whole cycle, one proton moves from the cytoplasm to the extracellular medium driven by one photon. These proton transfer reactions are, in principle, driven by successive proton affinity changes (conventionally expressed hy pK^sub a^) of the Schiff base, Asp85, the PRG and Asp96. The pathways of proton transfer between the residues have been discussed from the point of view of accessibility changes in the Schiff base (5,6), and have also recently been investigated by using QM/MM calculations for the L-to-M transition (7-9) and for the M-to-N transition (10). Here, on the basis of our FTIR studies and other data in the literature, we discuss our view of what regulates the affinity changes of the Schiff base and Asp85.

A WATER CLUSTER ON THE CYTOPLASMIC SIDE STABILIZES L RELATIVE TO M

In pH titrations of BR, the pK^sub a^ of the Schiff base in bacteriorhodopsin has been estimated to be ~13 (11), much higher than the value of ~7 for the retinal-butylamine complex in water-methanol solution (12), a model Schiff base. A solid state ^sup 15^N-NMR study on bacteriorhodopsin, in which the Schiff base was labeled with ^sup 15^N, has indicated that this elevated pK^sub a^ is caused by a directly interacting water molecule (Water402) and the negative charge of the counterion (Asp85) delocalized through a hydrogen bonding complex composed of the polar residues and additional water molecules (13) (Water401, Water406, Arg82 and Asp212) (Fig. 1). Lowering the pH of BR below 3 changes its color from purple to blue. This change results from the protonation of Asp85, indicating that the pK^sub a^ of Asp85 in BR is 3 (14).

The effect of light on bacteriorhodopsin is exerted primarily through the trans-to-cis isomerization of the C^sub 13^ = C^sub 14^ bond of the chromophore retinal. Resonance Raman studies have shown that the L, M and N intermediates at ambient temperature have the chromophore in the 13-cis, 14-trans, 15-trans conformation (15,16), in which the N-H bond of the Schiff base should be oriented opposite to that in RR, facing the cytoplasmic side. The Schiff base, however, is protonated in the L and N states but deprotonated in M. Proton transfer from the Schiff base to Asp85 in the L [arrow right] M transition occurs even at pH 3. The presence of the unprotonated state of the Schiff base, even in the pH range lower than the pK^sub a^ of the model Schiff base, may be the result of the absence of water molecules interacting directly with the Schiff base in M. A crystallographic model of BR at 1.55 [Angstrom] resolution (2) shows two water molecules (Water501 and Water502 in Fig. 1) on the cytoplasmic side, but they are not near the Schiff base. In contrast, the protonation of the Schiff base in L and N may be the result of interactions with water molecules that stabilize the protonated form of the Schiff base.