The role of proline residues in the proton pump mechanism of bacteriorhodopsin is investigated by Fourier-transform infrared (FTIR) difference spectroscopy using [15N]-proline labelled bacteriorhodopsin. Due to the isotopic shifts of absorbance bands, proline vibrations can be assigned to bands at 1616 cm−1 and between 1460 cm−1 and 1400 cm−1. At 1616 cm−1 a decrease of an absorbance band is observed in the intermediates K, L, M and in the dark adapted state (DA). This absorbance change can be correlated to a light induced H-bond alteration of a X-Pro amide carbonyl group. Isotopic shifts of difference bands between 1450 cm−1 and 1400 cm−1 are detected in K and M but are not observed in L and DA. From these frequency shifts of proline vibrations it is concluded that two proline residues undergo structural changes of their X-Pro C-N peptide bonds during proton pumping. The results support the suggestion that the more flexible X-Pro C-N bonds are used as hinges for a functional important structural motion of the protein backbone.
