Nano- and microsecond time-resolved FTIR spectroscopy of the halorhodopsin photocycle.

Step-scan Fourier transform infrared spectroscopy with 50 ns time resolution was applied to the early stages of the photocycle of halorhodopsin (hR) for the temperature range 3-42 degrees C. Kinetic data analysis with global fitting revealed two distinct kinetic processes associated with relaxations of the early red-shifted photoproduct hK; these processes have time constants tau 1 approximately equal to 280 ns and tau 2 approximately equal to 360 microns at 20 degrees C. Spectral features demonstrate that the tau 1 process corresponds to a transition between two distinct bathointermediates, hKE and hKL. The vibrational difference bands associated with both tau 1 and tau 2 transitions are spread throughout the whole 1800-900 cm-1 range. However, the largest bands correspond to ethylenic C=C stretches, fingerprint C-C stretches and hydrogen out-of-plane (HOOP) wags of the retinal chromophore. The time evolution of these difference bands indicate that both the tau 1 and tau 2 decay processes involve principally a relaxation of the chromophore and its immediate environment. The decay of the intense HOOP vibrations is nearly equally divided between the tau 1 and tau 2 processes, indicating a complex chromophore relaxation from a twisted nonrelaxed conformation in the primary (hKE) bathointermediate, to a less-twisted structure in hKL, and finally to a roughly planar structure in the hypsochromically shifted hL intermediate. This conclusion is also supported by the unexpectedly large positive entropy of activation observed for the tau 1 process. The two relaxations from hKE to hL are largely analogous to corresponding relaxations (KE-->KL-->L) in the bacteriorhodopsin photocycle, except that the second step is slowed down by over 200-fold in hR.