Proline residues undergo structural changes during proton pumping in bacteriorhodopsin

The role of proline residues in the proton pump mechanism of bacteriorhodopsin is investigated by Fourier‐transform infrared (FTIR) difference spectroscopy using [ 15 N]‐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.