KINETIC-ANALYSIS OF TIME-RESOLVED INFRARED DIFFERENCE SPECTRA OF THE L-INTERMEDIATES AND M-INTERMEDIATES OF BACTERIORHODOPSIN

Infrared difference spectra with 4 cm-1 spectral resolution and 10-mu-s temporal resolution, obtained previously with a stroboscopic Fourier-transform difference technique (Braiman et al., 1991, Proc. Natl. Acad. Sci. USA 88, 2388), were analyzed by means of a global exponential fitting procedure based on singular value decomposition. Using a simple linear kinetic model K --> L --> M for the bacteriorhodopsin (bR) photocycle in the time range 10-1000-mu-s at 16.5-degrees-C, it was possible to generate bR --> L and bR --> M difference spectra with signal/noise ratios comparable to those obtainable with low temperature difference spectroscopy. The resulting time-resolved bR --> L and bR --> M difference spectra are both very similar to the corresponding static FTIR difference spectra obtained at 175 K and 250 K, respectively. In the bR --> L spectrum, however, there are interesting differences that may indicate a greater degree of deprotonation of Asp-96 when L is formed at physiological temperatures than when it is observed in a low-temperature steady state.