Fourier transform infrared study of the N intermediate of bacteriorhodopsin

Visible absorption spectroscopic experiments show that the N intermediate is the main photoproduct of a highly hydrated film of the light-adapted bacteriorhodopsin (70% water by weight) at pH 10 and 274 K. The difference Fourier transform infrared spectrum between the N intermediate and unphotolyzed light-adapted bacteriorhodopsin was recorded under these conditions. A small amount of the M intermediate present did not affect this spectrum significantly. The difference spectrum exhibited a positive band at 1755 cm-1 (probably due to Asp-85) and a negative band at 1742 cm-1 (due to Asp-96), neither of which was observed for the M intermediate. The spectrum of the N intermediate at pH 7 was nearly identical with that at pH 10. Spectra at pH 10 also were measured with isotope-substituted samples. A vibrational band at 1692 cm-1 due to the peptide bond disappeared, and a band at 1558 cm-1 emerged upon formation of the N intermediate. The spectrum also displayed bands containing the N-H and C15-H in-plane bending vibrational modes at 1394 and 1303 cm-1. These frequencies are similar to those of the L intermediate while the intensities of these bands are larger than those in the L intermediate, suggesting that the Schiff bases of both the L and N intermediates have a strong hydrogen-bonding interaction with the protein and that the C12-H to C15-H region of the chromophore is less twisted in the N intermediate than in the L intermediate.