Rhodopsin Raman spectroscopy

The reaction sequence of Eq. 23-37 can be slowed by lowering the temperature. Thus, at 70K illumination of rhodopsin leads to a photostationary state in which only rhodopsin, bathorhodopsin, and a third form, isorhodopsin, are present in a constant ratio.510 Isorhodopsin (maximum absorption at 483 nm)513 contains 9-ds-retinal and is not on the pathway of Eq. 23-37. Resonance Raman spectroscopy at low temperature supports a distorted all-frans structure for the retinal Schiff base in bathorhodopsin.510 The same technique suggests the trans geometry of the C = N bond shown in Eqs. 23-38 and 23-39. Simple Schiff bases of 11-cz s-retinal undergo isomerization just as rapidly as does rhodopsin.514... 

Resonance Raman and NMR Studies. The major support to the protonation hypothesis is presently based on the recent application of resonance-Raman spectroscopy. (For recent reviews, see refs. 217-219.) The method uses an incident beam which is in resonance with the absorption of the retinyl chromophore. This results in the selective enhancement of the Raman cross sections coupled with the chromophore, relative to the very weak, non-resonant, modes of the opsin. Characteristic spectra are shown in Fig. 6. Early evidence for protonation came from the observation of a close similarity between the C=N vibrational frequency in rhodopsin and in a model protonated Schiff base (220). More conclusive arguments were provided by Oseroff and Callender, who carried out experiments at low temperatures in order to control sample photoability (221). It was observed that deuteration shifts the C=N vibration frequency from 1655 cm- to 1630 cm-- -, both in the pigment and in a model protonated Schiff base. 

Time-resolved Raman spectroscopy has proved to be a very useful tool to elucidate fast processes in biological molecules, for instance, to follow the fast structural changes during the visual process where, after photoexcitation of rhodopsin molecules, a sequence of energy transfer processes involving isomerization and proton transfer takes place. This subject is treated in more detail in Chap. 6 in comparison with other time-resolved techniques. 

Raman spectroscopy has been particularly useful in studies of rhodopsin and bacteriorhodopsin. As discussed in Chap. 4, excitatirai of rhodopsin or bacteriorho-dopsin by light causes isomerization of the retinyl chromophore. In rhodopsin, the chromophore changes from W-cis to all-trans in bacteriorhodopsin, it goes from dA -trans to 13-cA. Resonance Raman measurements showed that the isomerization is essentially complete in metastable intermediate states that form within a few ps [32-38]. The conformations of these states were ascertained by comparisons of the resonance Raman spectra with those of model compounds. 

Time-resolved Raman spectroscopy plays an important role in the investigations of molecular parameters in short-lived transition states [8.74]. In particular many fast structural changes in biological molecules during chemical reactions have been essentially disclosed by Raman spectroscopy. Examples are the conformational changes of rhodopsin during the visual process or the primary processes during photosynthesis (Sect. 15.6). 

Using a rapid flow technique, where a jet of molecules solved in a liquid flows through a focused laser beam, it is possible to study also photolabile molecules, such as rhodopsin, by resonance Raman spectroscopy [14.32]. This allows investigation of the interesting problem about the molecular mechanisms and dynamics of visual excitation. Rhodopsin molecules act as photoreceptors in the retina of vertebrates. If the rhodopsin sample is rapidly flowed through the focused laser beam, the fraction of isomerized molecules within the illuminated region stays small. This allows measurement of the resonance... 

The spectra of these intermediates can be obtained by time-resolved resonance Raman spectroscopy. An example is 1umirhodopsin spectroscopy. First a high-intensity pump pulse is used to initiate the bleaching of rhodopsin and, after a few ns, a second, low intensity probe pulse generates the Raman scattering from lumirhodopsin [14.32]. Many more examples of resonance Raman spectroscopy of biological molecules can be found in [14.33]. 

Oseroff, A.R. and Callender, R.H., Resonance Raman spectroscopy of rhodopsin in retinal disk... 

Spectroscopy and Physical Chemistry of Retinal and Visual Pigments. Several reviews and symposium proceedings discuss the spectroscopic, photochemical, or physicochemical properties of retinal and related compounds, and of natural and model visual pigments derived from them. " " In addition, many papers have been published dealing with specific aspects of the spectroscopy (u.v., n.m.r., resonance Raman) of retinals and rhodopsins" or with aspects of the photochemistry and physical chemistry of retinal derivatives which may be relevant to the functioning of rhodopsin and other visual pigments. The bacterial purple... 

This chapter has gathered together the current understanding of retinal photoisomerization in visual and archaeal rhodopsins mainly from the experimental point of view. Extensive studies by means of ultrafast spectroscopy of visual and archaeal rhodopsins have provided an answer to the question, What is the primary reaction in vision We now know that it is isomerization from 11-cis to all-trans form in visual rhodopsins and from all-trans to 13-cis form in archaeal rho-dopsin. Femtosecond spectroscopy of visual and archaeal rhodopsins eventually captured their excited states and, as a consequence, we now know that this unique photochemistry takes place in our eyes and in archaea. Such unique reactions are facilitated in the protein environment, and recent structural determinations have further improved our understanding on the basis of structure. In parallel, vibrational analysis of primary intermediates, such as resonance Raman and infrared spectroscopies, have provided insight into the isomerization mechanism. 

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... 

Infrared spectroscopy (IR) has not been extensively used in retinoid analysis (17,18). However, the newer techniques of Resonance Raman and infrared-difference spectroscopy have been applied to retinal proteins m rhodopsin and bacteriorhodopsin. By use of these techniques, it is possible to determine the structures of the chromophores m the visual pigments and in the intermediates of their photoreactions Also, it is possible to study the interactions between the chromophores and the protein, and the structural changes evoked in the protein by the photoreaction (1,19). 

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