Nano- and Microsecond Time-Resolved FTIR Spectroscopy of the Halorhodopsin Photocycle

• Published in: Photochemistry and photobiology Vol. 66; no. 6; pp. 755 - 763
• Main Authors: Dioumaev, Andrei K., Braiman, Mark S.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-12-1997
• Subjects: Bacteriorhodopsins - chemistry; Halorhodopsins; Kinetics; Photochemistry; Spectroscopy, Fourier Transform Infrared; Temperature; Thermodynamics
• ISSN: 0031-8655; 1751-1097
• DOI: 10.1111/j.1751-1097.1997.tb03220.x

— 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 T1⋍ 280 ns and T2⋍ 360 μs at 20°C. Spectral features demonstrate that the T1 process corresponds to a transition between two distinct bathointermediates, hKE and hKL. The vibrational difference bands associated with both T1 and T2 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 T1 and T2 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 T1 and T2 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 T1 process. The two relaxations from hKE to hL are largely analogous to corresponding relaxations (KE→ KL→ L) in the bacterior‐hodopsin photocycle, except that the second step is slowed down by over 200‐fold in hR.