Time-resolved resonance Raman spectroscopy of sensory rhodopsin II in the micro- and millisecond time range using gated cw excitation

A time‐resolved resonance Raman (RR) spectroscopic approach is presented that allows probing the dynamics of photoconvertible systems in a wide dynamics range covering more than six decades. It is based on an external opto‐electronic gating of continuous wave (cw) pump and probe lasers. Pump and probe events, the delay time as well as the repetition of this sequence can be varied from the nanosecond to the second time scale and thus adjusted to the desired time‐resolution and the recovery of the photoreversible system. Upon combination with the rotating cell technique, the approach offers several additional advantages, particularly for studying biological photoreceptors. Unlike in capillary flow systems, only small amounts of sample (ca 10 nmol) are required and no harmful mechanical stress is exerted on the proteins. External gating of cw lasers does not only provide freely adjustable pump and probe times but also avoids high pulse energies with pulsed laser excitation, thereby reducing the risk of unwanted photoinduced processes. The high potential of this approach is demonstrated by studying the formation and decay of a long‐lived intermediate (M400) of the sensory photoreceptor NpSRII from Natronobacterium pharaonis. The results are in very good agreement with the kinetic data derived from transient UV–vis absorption spectroscopy and demonstrate that this technique represents a powerful tool for studying the cofactor dynamics of biological photoreceptors in general. Copyright © 2006 John Wiley & Sons, Ltd.