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) similar or equal to 280 ns and tau(2) similar or equal to 360 mu s at 20 degrees C. Spectral features demonstrate that the tau(1) process corresponds to a transition between two distinct bathointermediates, hK(E) and hK(L). 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 (hK(E)) bathointermediate, to a less-twisted structure in hK(L), 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 hK(E) to hL are largely analogous to corresponding relaxations (K-E --> K-L --> L) in the bacteriorhodopsin photocycle, except that the second step is slowed down by over 200-fold in hR.