The electron shuttle heme protein Cyt-c(6) from the photosynthetic cyanobacterium Nostoc sp. PCC 7119 was immobilized on nanostructured Ag electrodes coated with SAMs that mimic different possible interactions with its natural reaction partner PSI. The structure, redox potential, and electron-transfer dynamics of the SAM-Cyt-c(6) complexes were investigated by TR-SERR spectroelectrochemistry. It is shown that the heterogeneous electron-transfer process is gated both in electrostatic and hydrophobic-hydrophilic complexes. At long tunneling distances, the reaction rate is controlled by the tunneling probability, while at shorter distances or higher driving forces, protein dynamics becomes the rate-limiting event.

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 (M 400 ) 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.

a b s t r a c tThe photocycle of the light-activated channel, channelrhodopsin-2 C128T, has been studied by resonance Raman (RR) spectroscopy focussing on the intermediates P380 and P353 that constitute a side pathway in the recovery of the parent state. The P353 species displays a UV-vis absorption spectrum with a fine-structure reminiscent of the reduced-retro form of bacteriorhodopsin, whereas the respective RR spectra differ substantially. Instead, the RR spectra of the P380/P353 intermediate couple are closely related to that of a free retinal in the all-trans configuration. These findings imply that the parent state recovery via P380/P353 involves the transient hydrolysis and re-formation of the retinal-protein linkage.

This article describes a method, based on surface-enhanced resonance Raman (SERR) spectroscopy, for studying the reaction dynamics of photoreceptors immobilized on metal electrodes. Time resolution and fresh sample conditions are achieved by synchronizing the rotational and translational motion of a novel kinematic electrode with the duration and time delay between the pump and probe events. The power and sensitivity of the method is illustrated by studying the photocycle of the sensory photoreceptor NpSRII and its sensitivity to the applied electric field. The results are compared with time-resolved resonance Raman measurements in solution.

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