Time-resolved step-scan FTIR investigations on the M1 → M2 transition in the light-driven proton pump bacteriorhodopsin

Using time-resolved step-scan FTIR spectroscopy, it has become possible to identify two different M states in the 5 to 300 mu s time range, which clearly differ from the M-N State. The identification has become possible by measuring a pure BR-->KL difference spectrum at 100 ns, which can be used to correct for the contributions of this intermediate in the later difference spectra. The subtraction of the later corrected difference spectra thus represent L --> M-1, M-2 difference spectra with varying relative amount of the two M states. The comparison of these spectra clearly reveals differences in the amide-II spectral range, and possibly also in the amide-I range. From these observations it can be concluded that the two M states do not differ in the chromophore structure but in the protein structure.