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

Abstract

Resonance Raman spectra of an invertebrate rhodopsin are reported. The spectrum of squid acid metarhodopsin is compared with the spectra of model compounds of the retinylidene chromophore in the all-trans conformation. Correlations made between acid metarhodopsin and these crystalline model compounds with known x-ray structures indicate that the chromophore in this intermediate is an all-trans protonated Schiff base. The data suggest a mechanism for the red shift in rhodopsin. Three years ago this laboratory reported the resonance Raman spectrum of the retinylidene chromophore of a visual pigment in a digitonin extract of bovine rhodopsin (1). Subsequent work here (1-5) and elsewhere (6-8) has demonstrated the sensitivity of resonance Raman spectroscopy as a probe of the electronic and molecular structure of the retinylidene chromophore in rhodopsin and its photochemically produced thermal intermediates. The wealth of information obtained from these ground state spectra, when coupled with recent data on the excited states of the retinylidene chromophore (9-12), can provide insights into the primary photophysical and photochemical processes of vision.The principal problem in this field is understanding the information present in the resonance Raman spectra of rhodopsin and its intermediates. Our approach has been to conduct parallel Raman studies of model compounds (13) with conformations and chemical environments that mimic those of rhodopsin and its intermediates. We have obtained the resonance Raman spectrum of a metarhodopsin, and we report here similarities between the spectrum of this rhodopsin intermediate and the spectra of several model compounds studied. This initial success not only has determined the conformation of the retinylidene chromophore in squid acid metarhodopsin, but also has provided experimental criteria for evaluating the credibility of proposed mechanisms for the absorption red shift observed in rhodopsin.Various experimental techniques have been used to obtain the resonance Raman spectra of a number of different rhodopsins and their intermediates. The principal finding of these studies is that the Schiff base in rhodopsin is protonated and can be deuterated. In the spectrum reported by Lewis et al. (1) and in later studies on the eye of a live rabbit by Lewis Raman spectra on flowing samplesof rhodopsin. Alternatively, both Lewis et al. (2) and Oseroff and Callender (6) used continuous lasers with stationary samples of rhodopsin maintained at low temperature to overcome rhodopsin's photolability.On the other hand, use of invertebrate rhodopsin obviates the need for the more elaborate sample techniques that have been resorted to for vertebrate rhodopsin Raman work. The bleaching sequence of squid rhodopsin-unlike vertebrate rhodopsins-does not terminate with the detachment of the retinylidene chromophore from opsin (14). In squid rhodopsin the thermal intermediate sequence in an acidic medium terminates with acid metarhodopsin (15, 16). Because the absorption maxima of rhodops...

Time-resolved resonance Raman spectroscopy of bacteriorhodopsin on the millisecond timescale.

“…We first compare its spectrum with two published spectra of the protonated n-butylamine Schiff base of all-trans retinal (17,21). Our spectrum shows common peaks at 1009 and 1204 cm-, and one of our peaks at 1173 and 1190 cm-1 could correlate with the peak shown (17,21) to exist at 1190 or 1164 cm-', respectively. The C=C stretch has been lowered to 1532 cm-1 in accordance with the shift in the absorption maximum (19).…”

“…The retinal in bR570 is thought to be a protonated Schiff base (11) of all-trans retinal (29,30). We first compare its spectrum with two published spectra of the protonated n-butylamine Schiff base of all-trans retinal (17,21). Our spectrum shows common peaks at 1009 and 1204 cm-, and one of our peaks at 1173 and 1190 cm-1 could correlate with the peak shown (17,21) to exist at 1190 or 1164 cm-', respectively.…”

“…Resonance Raman spectroscopy has been used to probe the structure of bacteriorhodopsin (8)(9)(10)(11), rhodopsin (12)(13)(14)(15)(16)(17), and the chromophore of both species, isomers of retinal (18)(19)(20)21). The kinetics of the photochemical cycle of bacteriorhodopsin have been studied by flash photolytic methods.…”

“…Our spectrum of bR570, however, has notable differences. The most serious mismatch between the spectra of bR570 and the all-trans protonated Schiff bases (17,21) …”

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

“…Second, selective resonance enhancement permits the assignment of particular Raman lines to specific molecules in the photostationary steady state (5). These experiments have already been used to examine the conformation and interactions of the retinal chromophore in rhodopsin, isorhodopsin, and several of their photolytic intermediates (5,(12)(13)(14)(15)(16). One objective of this study was to obtain a spectrum of bathorhodopsin that would provide information on the strength of the C=N bond at the C-15 position and on the degree of protonation of the Schiff base nitrogen.…”