ANION-PROTEIN INTERACTIONS DURING HALORHODOPSIN PUMPING - HALIDE BINDING AT THE PROTONATED SCHIFF-BASE

Halorhodopsin (hR), the light-driven chloride pump of Halobacterium ha lobium, has been studied by Fourier transform infrared (FTIR) spectros copy. Direct hydrogen bonding of halide ions with the protonated Schif f base (PSB) group was detected by means of halide-dependent perturbat ions 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-tem perature static difference spectroscopy at 1-cm(-1) resolution or a st roboscopic time-resolved technique with 5-ms temporal and 2-cm(-1) spe ctral 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 ty pe 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 f requency thus correlates with the strength of the hydrogen bond formed by the halide. Analogous halide-dependent shifts of the C=NH+ frequen cy were observed in IR spectra of model compound retinylidene PSB salt s. We also observed a significant halide dependence of the visible abs orption maximum of hR solubilized in lauryl maltoside detergent. From such halide perturbation effects, we conclude that there is a direct h ydrogen-bonded interaction between the Schiff base group and an extern ally supplied halide ion in the hR state. Halide perturbation effects are also observed for PSB-group vibrations in the hL state. Thus, desp ite an apparent overall weakening of hydrogen-bonding interactions of the PSB with its environment after chromophore photoisomerization to f orm 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 previous ly proposed two-site model.