Nanosecond time-resolved infrared spectroscopy distinguishes two K species in the bacteriorhodopsin photocycle

• Published in: Biophysical journal Vol. 68; no. 5; pp. 2073 - 2080
• Main Authors: Sasaki, J., Yuzawa, T., Kandori, H., Maeda, A., Hamaguchi, H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-05-1995
• Subjects: Bacteriorhodopsins - metabolism; Bacteriorhodopsins - radiation effects; Halobacterium - metabolism; Kinetics; Light; Mathematics; Models, Theoretical; Spectrophotometry, Infrared; Time Factors
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(95)80386-3

The photochemical reaction process of bacteriorhodopsin in the nanosecond time range (-120–860 ns) was measured in the 1400–900 cm-1 region with an improved time resolved dispersive-type infrared spectrometer. The system is equipped with a newly developed detection unit whose instrumental response to a 5-ns laser pulse has a full width of the half-maximum of 60 ns. It provides highly accurate data that enabled us to extract a kinetic process one order of magnitude faster than the instrumental response. The spectral changes in the 1400–900 cm-1 region were analyzed by singular value decomposition and resolved into three components. These components were separated by fitting with 10- and 1000-ns exponential functions and a step function, which were convoluted with the instrumental response function. The components with decay time constants of 10 and 1000 ns are named K and KL, respectively, on the basis of previous visible spectroscopy. The spectral shapes of K and KL are distinguishable by their hydrogen-out-of-plane (HOOP) modes, at 958 and 984 cm-1, respectively. The former corresponds to the K intermediate recorded at 77 K and the latter to a K-like photoproduct at 135 K. On the basis of published data, these bands are assigned to the 15-HOOP mode, indicating that the K and KL differ in a twist around the C14-C15 bond.