Resonance Raman studies of visual pigments.

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Ann. Rev. Biophys. Bioeng. /977. 6:33-55 Copyright (C) 1977 by Annual Reviews Tnc. All rights reserved

Annu. Rev. Biophys. Bioeng. 1977.6:33-55. Downloaded from www.annualreviews.org Access provided by University of Victoria on 11/15/15. For personal use only.

RESONANCE RAMAN STUDIES

.:-9086

OF VISUAL PIGMENTS Robert Callender Department of Physics. Cit y College of New York. New York. New York

10031

Barry Honig Department

of

Physical Chemistry. The Hebrew University. Jerusalem. Israel

INTRODUCTION Visual pigments of v etebr ates and many intervetebrates ar e co mposed of a small chromophore, II-cis retinal, covalently linked via a protonated Schiff base to a small protein called opsin. The pigments are situated in specialized membranes; the best studied, rhodopsin, is found in vetebrate rod o uter segments. The absor ption max ima of pigments range from about 430 to about 600 n m; the variation is due entirely to differences in the opsins. Light absorption by the v isual pigments initiates a series of events that leads to the excitation of the photoreceptor cell. The primary process in visual excitation elucidated by Wald and his colleagues (I) appears to be the photochemical izomerization of the chromophore to its all-trans conformation. This leads, via a yet unknown mechanism. to the release of a transmitter substance that subsequently decreases a current of sodium ions across the cell membrane (2). A considerable body of information exists on th e physical and chemical properties of visual pigments and the light-induced changes they undergo (for recent reviews see 3 and 4). Most progress has been based on spectroscopic and photochemical measurements and on theoretical studies. Recently. the application of resonance Raman spectroscopy has provided a signifi cant amount of important new structural information and has led to a more detailed description of visual pigments than has hitherto been possible. In the Raman effect, incident monochromatic radiation (usua lly produced by a laser) on a sa mp le is scattered and its f re quency is shifted by amounts corresponding to the normal mode frequencies of the material. Thus. the technique is complemen tary to infrared absorption in that it measures vibrational frequencies. However, it has some important advantages: measuring sample normal modes correspond ing to

33


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CALLENDER & HONIG

frequencies of 5-4000 cm-1 are possible aU at the same time with one apparatus; more information on selection rules is available from the experiment (in isotropic material, symmetric and asymmetric modes can be distinguished from depolariza tion rules); and water has al small Raman cross section and therefore generally does not give a troublesome background spectrum. In the resonance Raman case, the incident light frequency is in resonance with a particular absorption band. The cross-sections of Raman modes co upl ed to this absorption band are greatly enhanced. The Raman spectrum is then greatly simpli fied in that not all possible modes of the system are evidenced. For visual pigments, resonance Raman spectroscopy gives direct and specific information as to the vibra tional frequencies of the chromophore when the incident light used is in resonance with its absorption band. Since the vibrations of the protein are not seen, compli cated preparation procedures are not needed and, in fact, in situ experiments are possible. T he techni que has found rather wide application in th e study of other biologically interesting molecules (5, Sa). We divide this review into several sections. In the next section, a typical experi mental arrangement is described and methods for overcoming pro bl ems associated with Raman measurement of photolabile materials are discussed. The third section deals with interpreting Raman measurements, particularly concerning molecules related to the visible chromophore. Raman measurements on model compounds are then d escri bed. Fin al ly we discuss a number of impo rta nt prob lems of current interest pertaining to th(: spectroscopic and photochemical properties of visual pigments. Resonance Raman spectroscopy has provided a great deal of important information about these problems, and the relevant results are reviewed and future applications are considered.

EXPERIMENTAL CONFIGURATION AND THE PHOTOLABILITY PROBLEM The typi cal Raman apparatus consists of a laser, a s pectrometer, a photomultiplier tube light detector, and photon counting apparatus (Figure 1). Recently, small minicomputers have been interfaced to the photon-counting equipment and spec trometer, allowi ng increased flexibility and power in data storage and handling, as well as permitting the addition of separate spectral sweeps to increase signal to noise. The laser l ig ht is focused into the sample area, and the Raman scattered l ig ht is usually, but not necessarily, collected at 90°C from the incident beam. The electric field of the incident laser light is generally polarized, and the polarization properties of the scattered light can be determined by placing an analyzer in its path. It is not surprising that Raman spectroscopy has found widespread use with the advent of the laser. Its collimated monochromatic radiation of relatively high intensity per mits focusing its beam into a very small area. Thus a large fraction of the Raman scattered light can be collected by suitable optics. This is not possible with more conventional sources. It is im po rtan t to note that the Raman effect, even for resonance-enhanced cross-sections, is an extremely weak phenomenon. For example, it has been reported

RESONANCE RAMAN STUDIES OF VISUAL PIGMENTS

3S

PLOTTER SPECTROMETER MINI COMPUTER


PHOTON COUNTER

Figure 1

Schematic diagram of a Raman apparatus.

exciting and scattered light, respectively.

1)0

1)./

and 1)s are the frequency of the frequency of a sample normal

is the vibrational

mode.

(6) that the cross-section of the most intense Raman line of rhodopsin (the C=C ethylenic mode) measured with the S68.2-nm line o f the krypton laser is about 8 X 10-26 cm2/mol-sr. This is more than eight orders of m agnitude sm aller than the absorption cross-section at the same wavelength. In this particular case the rhodop sin molecule is more apt, by a factor of approximately \0+8, to absorb a photon than to scatter a Raman photon. Since visual pigments and many model systems are photosensitive in that the absorption of a photon changes the molecule, great care must be taken in performing Raman measurements. This consideration introduces a level of experimental complexity in obtaining spectra of well-characterized sam ples of model compounds and visual pigments that is not generally present in studies of other systems.

Photolability Consider, for example, a rhodopsin molecule in a focused laser beam of only a few milliwatts and at a wavelength near its absorption maximum. Within milliseconds or less (6, 7) the molecule absorbs a photon, which initiates a series of temperature dependent transformations of the pigment leading eventuaUy to the detachment of the aU-trans chromophore from the opsin (see below). Most o f the intermediates in this sequence can absorb a photon and revert back to rhodopsin or isorhodopsin (a photosensitive pigment formed from 9-cis retinal and opsin). The characteristic times for the formation of the various spectrally distinct, temperature-dependent species range from nanoseconds to seconds (4), whereas the characteristic time for the Raman event is lOBlonger than that for absorption and thus is on the order of days. It is clear that m any components are likely to be present in the beam, and thus the resulting spectra is difficult to interpret in terms of a single species. The problem o f sample photolability, however, is not insurmountable. Its effects can be controUed or minimized by the use of a number of experimental t echniques.

36

CALLENDER & HONIG


Stationary Measurements The early pioneering work of Rimai and his co-workers (8-15) used a conventional stationary configuration with the sample dissolved in solution and held in a cell or placed on filter paper. They noted that, in the case of experiments on retinal isomers, distinct spectra were obtained and so concluded that photoisomerization of the material had not occurred (11, 12). These experiments were performed with laser lines quite far from the absorption bands. Since the absorption cross-section de creases much more rapidly than the Raman cross-section, the effects of photolability were greatly reduced. Also, the molecules in solution are not stationary and move in and out of the laser beam by diffusion and, perhaps, convection currents (6). Thus, there is some opportunity for photoisomerized material in the focused laser beam to leave the beam and for new molecules, previously uneffected by light, to take their place. Nevertheless, the observation of separate spectr a for different retinal isomers is not a comple tely s atisf acto ry assay of the sample compositi on since a partial photoisomerization would also give distinct spectra but erroneous Raman results. I t has been recently shown, however, that the experiments of Rimai and his co workers, at least in the case of t he retinal isomers, are quite accurate, complicated by only a small degree of partial photoisomerization (6). On the other hand, an early study (16) of digitonin extracts of bovine rhodopsin, unfortunately, w as complicated by problems of photolability. Apparently in this case the exciting laser irradiation wavelength, although far from Ama" was suffi ciently close to induce major light-induced changes. In addition, the mitigating effects of molecular diffusion and convention were smaller than for retinals since rhodopsin is a larger molecule. The assay in this experiment was to measure the bulk absorption of the sampk before and after irradiation with laser light, and this was found not to change. Since the laser light w as focused and, therefore, could affect only the very small portilon of the sample actuaUy in the beam, changes in the bulk absorption would not be expected even though sample in the laser beam contains a mixture of photoproducts. The first experiment to control the effect of photolability of visual pigments was that of Oseroff & Callender ( 17) in their study of bovine rod outer segment vesicles at liquid nitrogen temp(:ratures. In this case, irradiation of bovine rhodopsin pro duces a sample mixture of rhodopsin, isorhodopsin, and bathorhodopsin (the com mon photoproduct of both rhodopsin and isorhodopsin; see below). Since these are interconvertible by light and since subsequent dark reactions are prevented at this low temperature, the composition of the sample rapidly (when using Jaser light) becomes photostationary and depends only on the w avelength of the exciting light. The exact sample composition could be easily assayed by using well-developed techniques of UV/visible absorption spectroscopy. To assign spectral bands to a particular species, a se(;ond pump laser beam was simultaneously applied to the sample. The function of the . pump beam was to modify the c omposi tio n of the sample while Raman spectra were obtained from the first probe beam. In this way all factors (including the resonance enhancement factors for various Raman lines, which are certainly wavelength dependent), except for sample composition, were held constant. The Raman bands could then be assigned to a particular s pecies.


37


Flow Measurements

A second technique which eliminates problems associated with photolability for samples in solution, was independently developed by Mathies et al (7) and Callender et al (6). This technique involves flowing solution samples contained either in a jet stream (7) or a capillary tube (6) through the laser beam with a velocity sufficient to insure that any given molecule has a low probability of absorbing a photon. The i dea behind this technique can be readily understood. A typical molecule moves across a square laser beam (for simplicity) in a time ro/v, where ro is the beam width and v is the molecular velocity transverse to the laser beam. While in the laser beam, the molecule is absorbing photons at a rate (photons per second) given Iofj Alr2, where 10 is the laser intensity (photons per square second) and fj A is the o absorption cross section (square centimeters per molecule). Thus, this molecule absorbs a total number of photons given by the product of these two numbers, i.e. Iofj Air 0 v. If this number is much less than one, our typical molecule has a rare chance of abs orbi ng a photon, implying that nearly all the sample in the laser beam is pure starting material. As has been shown (6, 7), the experimental parameters, Io,fj A,r and v, can be judiciously chosen so that this is the case. This does not mean that the sample is not absorbing photons and photoisomerizing. The fraction of molecules that do absorb are simply removed from the sample area and effectively replaced by new sample. In technique, it is very similar to the common use of flow ing systems in dye laser technology and rotating cells of Raman spectrometers, where the heated sample is removed from the laser beam being replaced by cooler material. A more detailed analysis of the flow techniques appears in other arti cles (6, 7). 0'

THEORETICAL ASPECTS OF THE RAMAN SPECTRA OF POLYENES A complete characterization of the R aman spectrum of a particular molecule re quires a normal mode analysis and a quantitative determi nation of the Raman intensity for each vibration and its dependence on the frequency of the exciting light. Unfortunately, it has been difficult to achieve a detailed theoretical description of Raman intensities, particularly for large molecules. First, a normal mode analysis of large molecules, especi ally those involving delocalized electrons as is the case for polyenes, is a considerable undertaking. Second, as discussed below, Raman intensi ties are dependent on excited state as well as ground state normal modes, and calculations of excited state potential surfaces are s ubj ect to considerable uncer tainty. Finally, the expression for Raman intensities involves sums over a complete set of both electronic and vibrational states. The extent to which these can be reduced to a few terms has not been studied in detail until recently. The difficulties associated with a complete theoretical analysis is perhaps the major drawback of the Raman technique since the data are often difficult to i nterpret in detailed molecular terms. In the past few years considerable theoretical effort has been directed towards interpreting the Raman spectra of large conj ugated molecules and significant

CALLENDER & HONIG

38

progress has been made (18 -24). In this section we discuss the factors that determine Raman intensities, particularly for polyenes, and outline the major theoretical prob lems currently under investigation. A brief discussion of the factors that determine .. their absorption spectra of polyenes is included.


Raman Intensities

The basic theory of Raman scattering developed by Placzek (25) was first applied in detail to molecular systems by Albrecht (26) and has since been both extensively extended and modified (1 8, 22-24). Experimental applications to biological systems have been previously reviewed (5, 5a) and new theoretical work is discussed in this volume by Warshel (24). The Raman Intensity Igl)-7gl for the transition from the lowest vibrational state i = 0 to the vibrational state i = 1 , both in the ground electronic state g, is proportional to the square of the term a (U/) of the polari zability tensor given by

�:l

pu 80,gl (v) I :: � e,j

Il
-140°C

LUMIRHODOPSIN (497nm) - 1O-55ec

)_400C


METARHODOPSIN I (47·8 -10-�ec

�

hv

!It(

>-lSoc

'

hv

METARHODoPsINlL(380nm) 2 -10 5.C

!

>OoC

ALL-TRANS �ETlNAL(387nm) + OPSIN (485 nm)ISORHODOPSIN

RHODOPSIN (498nm) Figure 6

The bleaching sequence of rhodopsin. Rhodopsin and isorhodopsin are placed

lowest in the figure to indicate they have lower free energy than their common photoproducts.

of considerable intrinsic interest. Raman experiments in visual pigments (see Figure 7), which have been d irected primarily at probing the chromophore, have d eter mined the nature of the chromophore-protein bond,have yielded important insights as to the mechanism of wavelength regulation of pigment absorption maxima, and have provided considerable data that may be used to characterize the light- and temper ature-ind uc ed changes that accompany the bleaching process. These and r elated probl e ms are considered in detail in the following sections. Retinal-Opsin Linkage

A lthough it had been suspected for some time that the Schiff base formed between retinal and the E-amino group of a lysine in opsin was protonated (3), a direct measurement became possible only through the use of Raman spectra (9). Lewis, Pager & Abrahamson (16) noted that the C=N vibrational frequency observed in their measurement of rhodopsin was close to that of model protonated Schiff bases and suggested, on this basis, that the Schiff base linkage in rhodopsin was proto nated. This result was also obtained in the experiments of Oseroff & Callender (17), which were performed at a low temperature to control sam ple photolability. These


A.

RHODOPS I N

\J

..

i

!) >-

0 = :s 0

B.

iii

ISORHODOPS I N

�

�

.... iii Z ... ....

�

Z "' � < III:

III 0 �

0

�

C.

800

RHODOPSIN

23�

I SORHODOPSIN

24�

BATHORHODOPSIN

53�

1000

1200

�,r-�

�

1400

�

1600 cm-l

Figure 7 Resonance Raman spectra of rhodopsin, isorhodopsin, and bathorhodopsin. [Re printed with permission from (0) Callender et al (6); (b and c) Oseroff & Callender (17).] Rhodopsin spectrum was taken at room temperature. Data in (b) and (c) were taken at liquid nitrogen temperature. Both the rhodopsin and isorhodopsin spectra are essentially identical to those measured at room temperature by Mathies, Oseroff & Stryer (7).



49

results by themselves, however, are not conclusive since the C=N stretching fre quency is likely to be sensitive to factors other than protonation. More conclusive evidence was provided by the measurements of Oseroff & Callender ( 17) who observed that in deuterated solutions the C=N vibration moves from about 1655 em-I (Figure 7) to about 1630 em-I, both in the pigments and for an isolated protonated Schiff base. Assuming that CH=NH+ vibration is decoupled from the rest of the molecule, the frequency shift can be understood entirely in terms of an increase in the reduced mass of the oscillator upon deuteration. Since no other normal vibration was significantly affected by deuteration, this result proves that the C=N bond is protonated. An interesting protein that uses light to actively transport protons though the purple membrane of Halobacterium halobium has been extensively characterized by Stoeckenius and co-workers (50). Due to its similarities to visual pigments (it has all-trans retinal as a chromophore and undergoes similar photochemically induced spectral changes), it has sometimes been called bacteriorhodopsin. A dditio na l simi larities are based on the Raman studies of Lewis et al (51) and Mendelsohn et al (52-54). Lewis et al (51) showed that the Schiff base linkage between the chromo phore and protein is protonated in a deuteration study, paralleling the work on rhodopsin. Recently this has been confirmed in a denaturation experiment (54). The purple membrane undergoes a light-driven reaction cycle that involves an intermedi ate absorbing at 412 nm (SO). The Schiff base line of this form is at 1622 cm-I, indicating that it is unprotonated (51). Thus the Raman results may suggest that the site of the Schiff base plays an active role in proton migration (5 I). The Colors of Visual Pigments

As first pointed out by Kropf & Hubbard (55) when the Schiff base of retinal is protonated, a positive charge is partially delocalized throughout the 11" electron system (29, 30). The net effect is an increase in electron delocalization and a corresponding decrease in bond alternation. The latter effect can be seen in X-ray studies of Schiff bases (Hamanaka and Mitsui, personal communication) as well as from the frequency shift of the C=C stretching vibrations considered above (13). The bathochromic shift that occurs upon protonation can be qualitatively under stood in terms of increased electron delocalization (see above). Moreover, for proto nated species, any mechanism that further increases electron delocalization will cause further red shifts (30). This follows from simple considerations of 11" electron theory and requires that a correlation exists between the C=C stretching frequency, v(C=C), and Amax> among various visual pigments (3, 9). Thus, the values of Amax = 450 nm, v(C=C) = 1560 cm-I for protonated Schiff bases ( 13, 17; Figure 5), Amax 498 nm, v(C=C) 1545 cm-I for rhodopsin (6, 7; Figure 7a), and Amax 543 nm, v(C=C) = 1539 cm-I for bathorhodopsin ( 17; Figure 7c) present a consistent and not unexpected trend. It is interesting that the C=C frequencies of th e 570- and 412-nm forms of the purple membrane of 1533 em-I and 1571 em-I, respectively (51), show the same type of correlation. It appears then that the mechanism by which the protein determines the absorp tion maxima of visual pigments involves regulating the degree of delocalization of =

=

=


50

CALLENDER & HONIG

the chromophores 1T electrons. As discussed above, the excited states of polyenes always involve considerable 1T electron delocalization and decreased bond alterna tion. Thus, as the Amax of a pigment increases with a concomitant decrease of bond alternation in the ground state, the equilibrium geometries of the ground and excited states approach one another (3�, 56). This explains the observed narrowing of pigment absorption bands as Amax shifts to the red since fewer lines in the C=C vibrational progression are Franck-Condon-allowed when the geometry shift is small (56). Since Raman intensities also depend on geometry changes, the same mechanism can account for a sensitivity of Raman intensities to Amax of the pig ment and for related differences between rhodopsin and protonated Schiff bases (39, 40). While the general principles concerning wavelength regulation of visual pigments now seem understood, the specific chromophore-protein interactions that determine the extent of 7T electron delocalization are less certain (for a recent discussion see reference 30). It appears that the most plausible specific model of chromophore protein interactions is one in which a negative counter-ion is bound to the proto nated nitrogen and where additional negatively charged or polar groups are positioned by the protein in the vicinity of the ring of the chromophore. Changes in the position of these groups determines the absorption maxima of various pig ments. At present it is not possible to fully exploit the Raman data to construct specific models of the chromophore-protein interaction although, as discussed above, they clearly require models based on 7T electron delocalization. However, some qualita tive inferences can be made. For example, the fact the C=N vibrational frequency is far less sensitive than the C=C stretch to changes in Amax ( 17) suggests that whatever charged or polar groups influence absorption maxima they are likely to be relatively far removed from the C=N bond. Mathies et al (39, 40) have pointed out that the spectrum of the II-cis protonated Schiff base of retinals is remarkably similar to that of rhodopsin, suggesting that the ground state conformation of ii-cis retinal in rhodopsin is very similar to the conformation of the ii-cis proto nated Schiff base in solution. Thus, theories of color based on chromophore distor tion, in particular twisting about C=C double bonds, appear unlikely. The Configuration 0/ the Protein-Bound Chromophore

It is useful to consider the extent to which Raman data can be used to identify the structure of a particula,r chromophore when bound to the protein. As discussed above the region between 1 100 and 1400 cm-! (the fingerprint region) is particularly sensitive to isomeric form at least for model compounds. This also appears to be true for visual pigments. First, rhodopsin and isorhodopsin display significantly different spectral patterns in this region (compare Figure 7 a and 7 b). Second, as reported by Mathies et al (39, 40) there are close similarities between the spectrum of the II-cis protonated Schiff base and rhodopsin (compare Figures 5a and 7a) and between the 9-cis protonated Schiff base and isorhodopsin (compare Figures 5b and 7 b). Thus it appears possible to identify the conformation of the chromophore even when bound to the protein. This should be extremely useful in analyzing steps in the bleaching sequence and in the cycle of bacteriorhodopsin. In fact, Sulkes et a1



51

(56a) has reported that squid metarhodopsin has a similar Raman spectrum to the all-trans protonated Schiff base of retinal. Additional isomer specific features appear to be preserved in the protein-bound chromophore. For example, rhodopsin, I I -cis retinal in solution, and the I I -cis protonated Schiff base all display a splitting of the "" toto cm-I methyl stretching vibration whereas isorhodopsin and its 9-cis analogues do not. Callender et al (6) have interpreted the two bands at 998 and 1 0 1 5 cm-I in the rhodopsin spectra as implying that the conformation of the l l -cis chromophore in rhodopsin is 1 2-s trans. This argument was based on analogies to model compounds that show that the only time the doublet structure is observed is in solutions of I I -cis retinal. Solutions of t his isomer (as opposed to crystals w hich consist of 1 2-s-cis configura tion) (43, 44) are likely to contain a significant (41 , 49), but not necessarily predomi nant (57), percentage of I I-cis, 1 2-s-trans retinal. It is important to consider factors that might contributt:; to differences between the Raman spectra of visual pigments and model compounds. As discussed in the section d ealing with pigment color, changes in the d egree of 7T electron d elocaliza tion would be expected to affect line intensities and positions throughout the spec trum. 7T electron delocalization affects vibrational frequencies by modifying bond orders a nd affects intensities by determining the shift in equilibrium nuclear configu ration in going from the ground to the excited state. These considerations are valid for a chromophore of fixed geometry but variable hma A second way in which the protein could affect Raman spectra is by modifying ground or excited state geometries. Warshel, for example, has reported that a strained all-trans geometry would enhance lines that are weakly discernible in crystals of all-trans retinal (58). An indication of this effect can be seen in studies of model compounds (see Figure 4). Crystals of I I -CIS retinal have, for exam ple, a dense seri�s of lines between 750 and 900 em-I. Other isomers (particularly all-trans) show some structure in this region although there are fewer lines that appear at m uch lower intensity. Now it is likely that the appearance of these modes in II-cis retinal is correlated with the fact that it is the only isomer with a significant nonplanar distortion in its polyene chain. The appearance of lines in the region of 750-900 em-I (as in Figure 7c) in a pigment is not necessarily an indication that an I I -cis isomer is present, but rather that a particular isomer that is planar in solution is prevented by the opsin from assuming a planar geometry. ••

The Primary Photochemical Event

The primary event in vision (the photochemical formation of bathorhodopsin) has recently been the subject of some controversy (59-62). It was originally proposed by Hubbard & Kropf (63) and Yoshizawa & Wald (64) that bathorhodopsin is formed by a photochemical cis-trans isomerization. A number of workers have questioned this simple description on the basis of the extremely fast formation time of bathorhodopsin (59) « 6 psec); however this appears to be ample time for isomerization to occur (58, 62). Alternate models hav e included a deprotonation of the Schiff base nitrogen (60) or the loss of a proton from a m ethyl group at position five accompanied by a simultaneous shift of double bonds along the polyene chain (61).


52

CALLENDER & HONIG

The low-temperature Raman study of Oseroff & Callender ( 1 7) provided the first conformation-specific data on bathorhodopsin and thus provides an important basis for the discussion of various models. The outstanding feature of the bathorhodopsin spectrum (Figure 7c) is three low-frequency lines at 856, 877, and 920 em-I . Retinal isomers have only very weak scattering near 856 and 877 cm-I and evidently none near 920 em- I . This suggests that bathorhodopsin is not a simple all-trans speci es and has structural features that are not characteristic of retinal isomers in solutions. As discussed above, it is likely that Raman i ntensity around 750--900 cm-I is asso ciated with nonplanar distortions of the polyene chain. In fact Warshel (58 ) has reported on the basis of model calculations that strain in the chromophore can give rise to the enhanced Raman i ntensities observed in the bathorhodopsin spectrum. Th us, the Raman data suggests that bathorhodopsin has a highly distorted geome· try, probably due to the inability of its all·trans chromophore to fit the opsin binding site. This would be consistent with the high free energy of bathorhodopsin relative to that of rhodopsin, the difference being over 1 3 kcal (62). The data summarized in Figure 7 strongly imply that the C=N vibrational fre· quency of bathorhodopsin is close (within 8 cm-I ) to that of rhodopsin and iso rhodopsin. This observation essentially precludes any model for bathorhodopsin that i nvolves deprotonation of the nitrogen (60) or a saturation of the C=N double bond (6 1 ). The 1 539-cm-I line in bathorhodopsin almost certainly corresponds to the C=C vibration shifted to lower frequency in accordance with the correlation between Am.. and v (C=C) (17). In a recent publication (62) various models for the primary event have been critically discussed, and it was shown that the only possibility consistent with all experimental o bservations is that of a photochemical cis-trans isomerization. The strongest evidence favoring cis-trans isomerization and essentially precluding all others, found in the original demonstration of Hubbard & Kropf (63) and Yo shizawa & Wald (64), is that it is possible at low temperature to establish a photo equilibrium between rhodopsin (1 I -cis) and isorhodopsin (9-cis) via bathorhodopsin (or lumirhodopsin) as a common intermediate. It is difficult to rationalize this picture without assuming isomerization around the corresponding cis double bonds as primary steps both in rhodopsin and isorhodopsin. This requires that their common i ntermediat,e have a trans conformation (but not necessarily a planar one). Raman measurements have provided an important confirmation of these argu ments by proving that photoisomerization of the visual chromophore is possible at low temperature. This follows from the observation (7) that isorhodopsin formed photochemicalIy at 77°K from rhodopsin in the experiments of Oserolf & Callender ( 1 7) has an essentially i dentical Raman spectrum to isorhodopsin formed from regeneration at room temperature as measured by Mathies, Oseroff & Stryer (7).

PROSPECTS It is clear that resonance Raman studies give detailed and i mportant information regarding the chromophore of visual pigments. Much remains to be done. Experi mentally, Raman measurements of the bleaching i ntermediates of rhodopsin past



53

bathorhodopsin have not been published. A complete study of the purple membrane protein should provide detailed results on the configurational changes taking place in that important system. Also, it is clear that a systematic resonance Raman study of other visual systems, as for example squid rhodopsin and chicken iodopsin, when combined with information concerning the known difference in their visual cycle and absorption spectrum would be valuable. Theoretically, it will be necessary to predict both the vibrational structure as well as the resonance-enhanced cross sections with some precision. New theoretical techniques coupled with model com pound studies such as isotopic substitution and other modifications of retinal should greatly facilitate the interpretation of resonance Raman spectra. Further probing of the chromophore site can be accomplished by the study of retinal analogues regenerated with opsin to form artificial pigments. Using pulsed laser techniques, the kinetics of the visual bleaching sequence could be probed in greater detai l than presently available. Experiments on live a ni mals, e.g. pulse laser experiments on rabbits by Lewis (64a), are also possible although the photolability problem will greatly complicate interpretation. Resonance Raman studies to date have necessarily provided information only about the chromophore site and its i nteraction with the opsin. However, if following the photoisomerization the function of rhodopsin as a transducer involves part of the protein not intimately associated with the chromophore, pres ent experiments will not be capable of probing the mechanisms i nvolved. Future work using nonreso nance Raman experiments [one has already been attempted (65)] that probe the entire opsin molecule may help here, although the information obtained will be diluted since functionally important as well as unimportant opsin components are simultaneously measured. The development of UV-tunable lasers is likely to be extremely important in studying proteins. It would then be possible to select laser lines in resonance with specifi c amino acid residues and, thereby, individually moni tor them. ACKNOWLEDGMENTS

Many of the i deas discussed in this paper have evolved as a result of our collabora tion with our colleagues, A. Doukas, B. Aton, T. Ebrey, M. Ottolenghi, and K. Nakanishi. One of us (BH) thanks A. Warshel for enlightening discussions on theoretical aspects of Raman spectra. We thank R. Mathies, T. Freedman, and L. 'Stryer for sending a copy of their manuscript before publi cation and for permission to reproduce Figure 5. We thank A. Lewis for permission to quote his unpublished results. Financial help from the Cottrell Research Corporation grant, City Univer sity Research Award Program and the National Science Foundation (BMS 7503020) is greatfully acknowledged. This manuscript was written in part while one of us (BH) was a Visiting Associate Professor of Biophysics at the University of Illinois and was supported by U.S. Public Health Service Grant EYO-1323 .

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CALLENDER & HONIG


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Modeling the resonance Raman spectrum of a metarhodopsin: implications for the color of visual pigments.

Resonance Raman studies of hemerythrin-ligand complexes.

Resonance Raman studies of Rieske-type proteins.

Photointermediates of visual pigments.

Pinniped visual pigments.

Primary structures of chicken cone visual pigments: vertebrate rhodopsins have evolved out of cone visual pigments.

Letter: Resonance Raman studies of nitric oxide hemoglobin.

Molecular genetics of human visual pigments.

Resonance Raman and surface-enhanced resonance Raman spectroscopy of hypericin.

FTIR study of primate color visual pigments.

Ionochromic behavior of Grecko visual pigments.

The gecko visual pigments: a microspectrophotometric study.

Classifying bivalve larvae using shell pigments identified by Raman spectroscopy.

The red and green cone visual pigments of deuternomalous trichromacy.

Part III: The evolution of photopic visual pigments in fishes.

Spectral tuning of rhodopsin and visual cone pigments.

Visual pigments of bioluminescent and nonbioluminescent deep-sea fishes.

The purification and molecular weight estimation of teleost visual pigments.

Visual pigments of the american eel Anguilla rostrata.

Resonance Raman studies of the carbonmonoxy form of catalase. Evidence for and effects of phenolate ligation.

Resonance Raman studies and structure of a sulfide complex of methemerythrin.

Resonance IR: a coherent multidimensional analogue of resonance Raman.

Resonance Raman studies of bathorhodopsin: evidence for a protonated Schiff base linkage.

Vitamin A2-based visual pigments in fully terrestrial vertebrates.