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 twodifferent M states in the 5 to 300 μs time range, which clearly differ from the M N state.The identification has become possible by measuring a pure BR→KL differencespectrum at 100 ns, which can be used to correct for the contributions of thisintermediate in the later difference spectra. The subtraction of the later correcteddifference spectra thus represent L→M 1 , M 2 difference spectra with varying relativeamount of the two M states. The comparison of these spectra clearly reveals differencesin the amide-II spectral range, and possibly also in the amide-I range. From theseobservations it can be concluded that the two M states do not differ in the chromophorestructure but in the protein structure.