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° 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 τ1 ≃ 280 ns and τ2 ≃ 360 μs at 20°C. Spectral features demonstrate that the τ1 process corresponds to a transition between two distinct bathointermediates, hKE and hKL. The vibrational difference bands associated with both τ1 and τ2 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 τ1 and τ2 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 τ1 and τ2 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 τ1 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.
|Number of pages
|Photochemistry and photobiology
|Published - Dec 1997
ASJC Scopus subject areas
- Physical and Theoretical Chemistry