Two bathointermediates of the bacteriorhodopsin photocycle, distinguished by nanosecond time-resolved FTIR spectroscopy at room temperature

FTIR step-scan spectroscopy with 10 ns nominal time resolution was applied to the early stages of the photocycle of bacteriorhodopsin at room temperature. Kinetic data analysis with global fitting revealed two distinct bathointermediates prior to relaxation into the L state. The late bathointermediate (which we term K_L) is the K state as originally defined by Lozier et al. (Biophys. J. 1975, 15, 955). The earlier bathointermediate, K_E, decays to K_L with an apparent time constant of 640 ± 90 ns at 5 °C; this decay process is approximately 50-100 ns at 25 °C. The transient change in vibrational difference bands associated with this transition are spread throughout the 1800-800 cm^-1 range. However, the largest differences in the spectra of K_E and K_L appear to be mostly associated with a relaxation of the Schiff base end of the retinal chromophore and/or its immediate environment. Both our K_E and K_L IR spectra are different from the spectra of bathoproducts obtained at cryogenic temperatures. Our fitted time constants also imply that neither of these intermediates (K_E and K_L) can be identified with the K or KL states as defined by Shichida et al. (Biochim. Biophys. Acta 1983, 723, 240), and consequently, our spectra for K_E and K_L are markedly different from IR spectra calculated for those states by Weidlich and Siebert (Appl. Spectrosc. 1993, 47, 1394) and Sasaki et al. (Biophys. J. 1995, 68, 2073). That is, here we characterize a new nanosecond bathointermediate, K_E, in the bacteriorhodopsin photocycle under physiological conditions, which was not detected in previous studies.