Rhodopsin, picosecond spectroscopy

Example 2—The first stage in this process of vision has been the excitation of rhodopsin. Rhodopsin partially gets deactivated forming an intermediate, prelumirhodopsin or bathorhodopsin. Picosecond spectroscopy reveals that prelumirhodopsin gets formed because of an intramolecular proton transfer—a jump of a proton from one position to another. 

The kinetics of the photoisomerization of bilirubin has been studied because of the relevance to phototherapy. The fluorescence of bilirubin increases on binding to human serum albumin. This and other primary photoprocesses have been investigated by picosecond spectroscopy. Karvaly has put forward a new photochemical mechanism for energy conversion in bacteriorhodopsin. An extensive review of the photophysics of light transduction in rhodopsin and bacteriorhodopsin has been made by Birge. The dynamics of cis-trans isomerization in rhodopsin has been analysed by INDO-CISD molecular orbital theory. Similar calculations on polyenes and cyanine dyes have also been reported. A new picosecond resonance Raman technique shows that a distorted... 

The Photoactive Yellow Protein (PYP) is the blue-light photoreceptor that presumably mediates negative phototaxis of the purple bacterium Halorhodospira halophila [1]. Its chromophore is the deprotonated trans-p-coumaric acid covalently linked, via a thioester bond, to the unique cystein residue of the protein. Like for rhodopsins, the trans to cis isomerization of the chromophore was shown to be the first overall step of the PYP photocycle, but the reaction path that leads to the formation of the cis isomer is not clear yet (for review see [2]). From time-resolved spectroscopy measurements on native PYP in solution, it came out that the excited-state deactivation involves a series of fast events on the subpicosecond and picosecond timescales correlated to the chromophore reconfiguration [3-7]. On the other hand, chromophore H-bonding to the nearest amino acids was shown to play a key role in the trans excited state decay kinetics [3,8]. In an attempt to evaluate further the role of the mesoscopic environment in the photophysics of PYP, we made a comparative study of the native and denatured PYP. The excited-state relaxation path and kinetics were monitored by subpicosecond time-resolved absorption and gain spectroscopy. 

Figure 8. Ultrafast processes associated with bathorhodopsin and hypsorhodopsin monitored by picosecond absorption spectroscopy. (A) Absorbance changes (aA) as function of time monitored at 530 nm, showing the decay of a species (PBAT) identified as the precursor of BAT. (B) Arrhenius plot for the rate constant of this process in rhodopsin and in deuterated rhodopsin. (C) Decay of hypsorhodopsin. (D) Formation of bathorhodopsin. [(A) and (B) data from ref. 301 for bovine rhodopsin (in 0.1 M Ammonix LO, 66% ethylene glycol, at pH 7.0). (C) and (D) data from ref. 298 for squid rhodopsin in 2% digitonin (pH 10.5) at room temperature.]...

The Visual Transduction Process. Picosecond absorption spectroscopy which utilizes OMCDs also has provided important mechanistic information that previously was not available by means of other techniques. Detailed pathways of a number of reactions which are important from a physical, chemical, and/or biological viewpoint have been elucidated by means of this technique. A recent picosecond spectroscopic study by Spalink et. al. (30) has demonstrated that an experimental criterion, which has been used to support the hypothesis that cis-trans isomerization (31) is the primary event in the visual transduction process, is not true. This criterion is based on the commonly occurring statement that both the naturally occurring 11-cis-rhodopsin and the synthetic 9-cis-rhodopsin lead to the same primary photochemical product, bathorhodopsin. Of course, the existence of a common intermediate generated from either 11-cis- or 9-cis-rhodopsin would support the commonly proposed mechanism of cis-trans isomerization as the primary event in the visual transduction process. However, the data obtained by Spalink et. al. (30) indicate that a common intermediate is not generated from both rhodopsins. 

Intermolecular Photo-Induced Electron Transfer. Picosecond absorption spectroscopy has also been applied recently to studies of intermolecular electron transfer on the picosecond time scale.(34) As in the previously described study of rhodopsin, the photo-induced intermolecular electron transfer between chloranil (CHL) and the arenes, naphthalene (NAP), 9,10-dihydrophenanthrene (DHP), and indene (IN) was studied by means of picosecond absorption spectroscopy which utilizes an OMCD. Difference absorption spectra of samples of CHL and one of these particular arenes in acetonitrile were measured at selected delay times after excitation at 355-nm with 25-ps FWHM laser pulses. These picosecond spectroscopic studies revealed information about the mechanism of intermolecular electron transfer and subsequent radical ion formation that was not possible in previous spectroscopic studies performed on the nanosecond (35-37) and microsecond (38,39) time scales. 

In the case of 5-membered rhodopsin, only a long-lived excited state (r = 85 ps) was formed without any ground-state photoproduct (Fig. 4.5D), giving direct evidence that the CTI is the primary event in vision [39]. Excitation of 7-membered rhodopsin, on the other hand, yielded a ground-state photoproduct with a spectrum similar to photorhodopsin (Fig. 4.5C). These different results were interpreted in terms of the rotational flexibility along the C11-C12 double bond [39]. This hypothesis was further supported by the results with an 8-membered rhodopsin that possesses a more flexible ring. Upon excitation of 8-membered rhodopsin with a 21 ps pulse, two photoproducts - photorhodopsin-like and bathorhodopsin-like products - were observed (Fig. 4.5B) [40], Photorhodopsin is a precursor of bathorhodopsin found by picosecond transient absorption spectroscopy [41]. Thus, the picosecond absorption studies directly elucidated the correlation between the primary processes of rhodopsin and the flexibility of the Cl 1-02 double bond of the chromophore, and we eventually concluded that the respective potential surfaces were as shown in Fig. 4.5 [10,40]. 

Because the high quantum yield originates from the high-rate isomerization, which competes with other relaxation processes in the excited state of rhodopsin, ultrafast laser spectroscopies were applied to investigate the isomerization process of the retinal chromophore. Picosecond time-resolved spectroscopy was appHed to the photochemistry of rhodopsin, and the formation of the primary intermediates was reported, such as photorhodopsin and bathorhodopsin at room temperature. - - However, the time resolution needed to be improved in order to detect the cis-tram isomerization process in the excited state of rhodopsin. The direct observation of the rhodopsin excited state was reported in 1991, in which the primary intermediate photorhodopsin formed from the excited state of rhodopsin within 200 fs. Later, the effects of oscillatory features with a period of 550 fs (60 cm ) on the formation kinetics of photorhodopsin, were observed, suggesting that the primary step in vision is a vibrationally coherent process. 

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