Halide Binding by the D212N Mutant of Bacteriorhodopsin Affects Hydrogen Bonding of Water in the Active Site

Abstract

Bacteriorhodopsin (BR), a membrane protein found in Halobacterium salinarum, functions as a light-driven proton pump. The Schiff base region has a quadrupolar structure with positive charges located at the protonated Schiff base and Arg82, and the counterbalancing negative charges located at Asp85 and Asp212. The quadropole inside the protein is stabilized by three water molecules, forming a roughly planar pentagonal cluster composed of these waters and two oxygens of Asp85 and Asp212 (one from each carboxylate side chain). It is known that BR lacks proton-pumping activity if Asp85 or Asp212 is neutralized by mutation, but binding of Cl- has different functional effects in mutants at these positions. Binding of Cl- to D85T converts into a chloride ion pump (Sasaki, J., Brown, L. S., Chon, Y.-S., Kandori, H., Maeda, A., Needleman, R., and Lanyi, J. K. (1995) Science 269, 73-75). On the other hand, photovoltage measurements suggested that binding of Cl- to D212N restores the proton-pumping activity at low pH (Moltke, S., Krebs, M. P., Mollaaghababa, R., Khorana, H. G., and Heyn, M. P. (1995) Biophys. J. 69, 2074-2083). In this paper, we studied halide-bound D212N mutant BR in detail. Light-induced pH changes in a suspension of proteoliposomes containing D212N(Cl-) at pH 5 clearly showed that Cl- restores the proton-pumping activity. Spectral blue-shift induced by halide binding to D212N indicates that halides affect the counterion of the protonated Schiff base, whereas much smaller halide dependence of the lambdamax than in D85T suggests that the binding site is distant from the chromophore. In fact, the K minus BR difference Fourier-transform infrared (FTIR) spectra of D212N at 77 K exhibit little halide dependence for vibrational bands of retinal and protein. The only halide-dependent bands were the C=N stretch of Arg82 and some water O-D stretches, suggesting that these groups constitute a halide-binding pocket. A strongly hydrogen-bonded water molecule is observed for halide-bound D212N, but not for halide-free D212N, which is consistent with our hypothesis that such a water molecule is a prerequisite for proton-pumping activity of rhodopsins. We concluded that halide binding near Arg82 in D212N restores the water-containing hydrogen-bonding network in the Schiff base region. In particular, the ion pair formed by the Schiff base and Asp85 through a strongly hydrogen-bonded water is essential for the proton-pumping activity of this mutant and may be controlled by the halide binding to the distant site.

Hydrogen Bonds of Protein‐Bound Water Molecules in Rhodopsins

“…Figure 18(B)(b) also shows the absence of strongly hydrogen-bonded water molecules (<2400 cm À1 ) upon mutation of Asp212 to Asn [20,21]. Nevertheless, strongly hydrogen-bonded water molecules appear at 2350 2310 cm À1 when halide binds to the D212N mutant ( Figure 18.8(B)(c to e)) [47]. As these mutants pump protons, the results in Figure 18.8 are consistent with our hypothesis that strongly hydrogen-bonded water molecules are a prerequisite for proton-pumping activity of rhodopsins.…”

“…Figure 18.9 summarizes the results, which clearly show a strong correlation between the presence of strongly hydrogen-bonded water molecule(s) and proton-pumping activity. For example, strongly hydrogenbonded water molecules are observed for BR [19][20][21], D212N(Cl À ) BR [47], azide-bound HR [48], salinibacter SRI [49], SRII [50], proteorhodopsin [51,52] and Leptosphaeria rhodopsin [53], which all pump protons. Strongly hydrogen-bonded water molecules were not observed for D85S(Cl À ) BR [45], Figure 18.9 Various rhodopsins are classified in view of (i) proton-pump activity and (ii) whether they have strongly hydrogen-bonded water molecules (OÀD stretch in D 2 O at <2400 cm À1 ) (See Plate 22) HR [41,46], Anabaena sensory rhodopsin [54,55], Neurospora rhodopsin [56], bovine Rh [57] and squid Rh [58], which have no proton-pumping activity.…”

Protein‐Controlled Ultrafast Photoisomerization in Rhodopsin and Bacteriorhodopsin

“…Cl − -pumping D85S BR has no strongly hydrogen -bonded water molecules, 92 but proton -pumping D212N(Cl − ) BR has strongly hydrogen -bonded water molecules. 93 These results were interpreted in terms of the proposal that the presence of a strong hydrogen bond of water is prerequisite for proton pumping in Rhs. 83 It is likely that destabilization of a water -containing hydrogen -bonding network plays an important role for light -energy storage in this case.…”

Color Change of Proteorhodopsin by a Single Amino Acid Replacement at a Distant Cytoplasmic Loop

“…HPLC analysis was performed as described previously. [27] Protein stability was measured as described previously. [28] …”