The Mechanism of Photobiological Systems Studied by Time-Resolved Nanosecond Step-Scan FT-IR Spectroscopy

Abstract

The elucidation of the mechanism of photobiological systems by studying the molecuar alterations during their photoreactions is one of the key issues in biomolecular spectroscopy. It is important to monitor the light-induced molecular changes of both the chromophore and the protein. Time-resolved infrared difference spectroscopy has been shown to be a very useful tool for the study of such processes (e.g. [1]). Due to its non-selectivity, it is sensitive for both parts of these systems. Previously, we have shown that time-resolved step-scan FT-IR spectroscopy is very well suited for the sudy of such systems that can be triggered repetitively. The multiplex advantage is retained and very small spectral changes can be detected over a broad spectral range [2, 3]. In our earlier implementation, the time-resolution was limited by the rise-time of the detector and the acquisition electronics to 500 ns. This inhibited the measurements of early events in the photoreactions such as the K intermediates of bacteriorhodopsin and halorhodopsin. By replacing the photoconductive MCT detector by a photovoltaic one, and by incorporating a faster acquisition electronics (200 MHz transient recorder, 150 MHz amplifiers), the time-resolution could be increased to 50 ns. We have used this improved instrument to study the photoreactions of the proton pump bacteriorhodopsin [4, 5] (from Halobacterium salinarium) and of the chloride pump halorhodopsin [5, 6] (from Natronobacterium pharaonis). The measurement of the reactions at early times proved to be important for their elucidation at later times.