Infrared evidence that the Schiff base of bacteriorhodopsin is protonated: bR570 and K intermediates.

Abstract

It is possible, by using Fourier-transform IR (FTIR) difference spectroscopy, to detect the conformational changes occurring in both the protein and the chromophore of Halobacterium halobium bacteriorhodopsin during the photocycle. In contrast to Raman spectroscopy, a laser is unnecessary and hence the problem of a perturbing probe beam is eliminated. The relatively high signal-to-noise ratio obtainable with FTIR enables measurements to be made in minutes over a large spectral range. This method was used to examine the state of protonation of the retinylidene Schiff base in light-adapted bR570 and in K, the 1st intermediate in the photocycle. Resonance Raman spectroscopy provides evidence that bR570 is protonated, but these results have been questioned on theoretical and experimental grounds. FTIR difference spectral changes in the bR570-to-K transition clearly indicate that bR570 contains a protonated Schiff base. In contrast, the K intermediate displays a Schiff base that is altered but still is associated to some degree with a proton. Because the low-temperature FTIR difference spectrum of bR570 and K is similar to the recently reported low-temperature resonance Raman spectra of bR570 and K, most, but not all, vibrational changes in the bR570-to-K transition can be assigned to the chromophore. These results are consistent with a simple model of the 1st step in the photocycle which involves a movement of the Schiff base proton away from a counterion.