Halorhodopsin (hR), the light-driven chloride pump of Halobacterium halobium, has been studied by Fourier transform infrared (FTIR) spectroscopy. Direct hydrogen bonding of halide ions with the protonated Schiff base (PSB) group was detected by means of halide-dependent perturbations on this group's vibrational frequencies. FTIR difference spectra were obtained of the hR ⟶ hL photoreaction in reconstituted membrane vesicles. Nearly identical results were obtained using either low-temperature static difference spectroscopy at 1-cm−1 resolution or a stroboscopic time-resolved technique with 5-ms temporal and 2-cm−1 spectral resolution. The frequency of the negative difference band due to the PSB C=N stretch mode in the hR state shows a dependence on the type of halide counteranion that is present, 1632 cm−1 in the presence of Cl-, 1631 cm−1 in Br−, and 1629 cm−1 in I-. The C=NH+ stretch frequency thus correlates with the strength of the hydrogen bond formed by the halide. Analogous halide-dependent shifts of the C=NH+ frequency were observed in IR spectra of model compound retinylidene PSB salts. We also observed a significant halide dependence of the visible absorption maximum of hR solubilized in lauryl maltoside detergent. From such halide perturbation effects, we conclude that there is a direct hydrogen-bonded interaction between the Schiff base group and an externally supplied halide ion in the hR state. Halide perturbation effects are also observed for PSB-group vibrations in the hL state. Thus, despite an apparent overall weakening of hydrogen-bonding interactions of the PSB with its environment after chromophore photoisomerization to form hL, the PSB remains hydrogen-bonded to the halide. The results are best explained in terms of a “one-site, two-state” model for anion binding near the chromophore in the hR state, as opposed to a previously proposed two-site model.
|Original language||English (US)|
|Number of pages||10|
|State||Published - Feb 1 1994|
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