A method was developed to measure Fouriertransform infrared (FTIR) difference spectra of detergentsolubilized rhodopsin expressed in COS cells. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and three expressed rhodopsin mutants with amino acid replacements of membrane-embedded carboxylic acid groups: Asp-83 -* Asn (D83N), Gln (E122Q), and the double mutant D83N/E122Q. Each of the mutant opsins bound 11-cis-retinal to yield a visible light-absorbing pigment. Upon illumination, each of the mutant pigments formed a metarhodopsin fl-like species with maximal absorption at 380 nm that was able to activate guanine nucleotide exchange by btanducin. Rhodopsin versus metarhodopsin iH-like photoproduct FTIR-difference spectra were recorded for each sample. The COS-ceil rhodopsin and mutant difference spectra showed close correspondence to that of rhodopsin from disc membranes.Difference bands (rhodopsin/metarhodopsin II) at 1767/1750 cm'i and at 1734/1745 cm-' were absent from the spectra of mutants D83N and E122Q, respectively. Both bands were absent from the spectrum of the double mutant D83N/E122Q. These results show that Asp-83 and Glu-122 are protonated both in rhodopsin and in metarhodopsin H, in agreement with the isotope effects observed in spectra measured in 2H20. A photoproduct band at 1712 cm-' was not affected by either single or double replacements at positions 83 and 122. We deduce that the 1712 cm-' band arises from the protonation of Glu-113 in metarhodopsin II. Rhodopsin is a member of the superfamily of seventransmembrane-helix, G protein-coupled receptors. The rhodopsin chromophore 11-cis-retinal is covalently bound to the protein via a protonated Schiff base linkage (1) to a lysine residue (Lys-296 in bovine rhodopsin) (2, 3). After photoisomerization of the chromophore, thermal relaxation leads to an active conformation, R*, which binds the G protein transducin and thereby couples photon absorption to the visual signal transduction cascade. It has been shown by chemical modifications of Lys-296 in bovine rhodopsin that the deprotonation of the Schiff base is a prerequisite for R* formation (4, 5). Spectroscopically, this state is designated metarhodopsin II (MII) and characterized by a visible absorption maximum (Am.) at 380 nm, indicative of the unprotonated Schiff base of all-trans-retinal. Biochemical studies (6-10) and resonance Raman spectroscopy (11) of recombinant rhodopsins have shown that the positive charge at the Schiff base nitrogen in rhodopsin is stabilized by Glu-113, which acts as a Schiff base counterion in the transmembrane domain of the opsin.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.To investigate the protonation states and possible protonation changes of membrane-embedded carboxyl groups in rhodopsin and its MII photoproduct, we have performed Fourier-transform i...

In order to investigate the molecular mechanism of rhodopsin photoactivation, site-directed mutants of bovine rhodopsin were studied by Fourier-transform infrared (FTIR) difference spectroscopy. Rhodopsin mutants E113D and E113A were prepared in which the retinylidene Schiff base counterion, Glu113, was replaced by Asp and Ala, respectively. FTIR difference spectra were recorded and compared with spectra of recombinant native rhodopsin. Both mutant pigments formed photoproducts at 0 degrees C with vibrational absorption bands typical of the metarhodopsin II (MII) state of rhodopsin. The FTIR difference spectrum of E113D was nearly identical to that of rhodopsin. A positive band at 1712 cm-1 caused by the protonation of an internal carboxylic acid in rhodopsin was shifted slightly to 1709 cm-1 in mutant E113D. E113A was studied at acidic pH in the presence of chloride as an inorganic counterion to the protonated Schiff base. The 1712-cm-1 (1709-cm-1) band was absent in the FTIR difference spectrum of mutant E113A. Therefore, we have assigned the 1712-cm-1 absorbance band to the C = O stretching vibration of protonated Glu113 in MII of rhodopsin. These results show that the Schiff base counterion of rhodopsin, the carboxylate side chain of Glu113, becomes protonated during MII formation.

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Time-resolved vibrational spectra are used to elucidate the structural changes in the retinal chromophore within the K-590 intermediate that precedes the formation of the L-550 intermediate in the room-temperature (RT) bacteriorhodopsin (BR) photocycle. Measured by picosecond time-resolved coherent anti-Stokes Raman scattering (PTR/CARS), these vibrational data are recorded within the 750 cm-1 to 1720 cm-1 spectral region and with time delays of 50-260 ns after the RT/BR photocycle is optically initiated by pulsed (< 3 ps, 1.75 nJ) excitation. Although K-590 remains structurally unchanged throughout the 50-ps to 1-ns time interval, distinct structural changes do appear over the 1-ns to 260-ns period. Specifically, comparisons of the 50-ps PTR/CARS spectra with those recorded with time delays of 1 ns to 260 ns reveal 1) three types of changes in the hydrogen-out-of-plane (HOOP) region: the appearance of a strong, new feature at 984 cm-1; intensity decreases for the bands at 957 cm-1, 952 cm-1, and 939 cm-1; and small changes intensity and/or frequency of bands at 855 cm-1 and 805 cm-1; and 2) two types of changes in the C-C stretching region: the intensity increase in the band at 1196 cm-1 and small intensity changes and/or frequency shifts for bands at 1300 cm-1 and 1362 cm-1. No changes are observed in the C = C stretching region, and no bands assignable to the Schiff base stretching mode (C = NH+) mode are found in any of the PTR/CARS spectra assignable to K-590. These PTR/CARS data are used, together with vibrational mode assignments derived from previous work, to characterize the retinal structural changes in K-590 as it evolves from its 3.5-ps formation (ps/K-590) through the nanosecond time regime (ns/K-590) that precedes the formation of L-550. The PTR/CARS data suggest that changes in the torsional modes near the C14-C15 = N bonds are directly associated with the appearance of ns/K-590, and perhaps with the KL intermediate proposed in earlier studies. These vibrational data can be primarily interpreted in terms of the degree of twisting of the C14-C15 retinal bond. Such twisting may be accompanied by changes in the adjacent protein. Other smaller, but nonetheless clear, spectral changes indicate that alterations along the retinal polyene chain also occur. The changes in the retinal structure are preliminary to the deprotonation of the Schiff base nitrogen during the formation of M-412. The time constant for the ps/ns K-590 transformation is estimated from the amplitude change of four vibrational bands in the HOOP region to be 40-70 ns.

The vibrational spectrum (650-1750 cm(-1)) of the lumi-rhodopsin (lumi) intermediate formed in the microsecond time regime of the room-temperature rhodopsin (RhRT) photoreaction is measured for the first time using picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). The vibrational spectrum of lumi is recorded 2.5 micros after the 3-ps, 500-nm excitation of RhRT. Complementary to Fourier transform infrared spectra recorded at Rh sample temperatures low enough to freeze lumi, these PTR/CARS results provide the first detailed view of the vibrational degrees of freedom of room-temperature lumi (lumiRT) through the identification of 21 bands. The exceptionally low intensity (compared to those observed in bathoRT) of the hydrogen out-of-plane (HOOP) bands, the moderate intensity and absolute positions of C-C stretching bands, and the presence of high-intensity C==C stretching bands suggest that lumiRT contains an almost planar (nontwisting), all-trans retinal geometry. Independently, the 944-cm(-1) position of the most intense HOOP band implies that a resonance coupling exists between the out-of-plane retinal vibrations and at least one group among the amino acids comprising the retinal binding pocket. The formation of lumiRT, monitored via PTR/CARS spectra recorded on the nanosecond time scale, can be associated with the decay of the blue-shifted intermediate (BSI(RT)) formed in equilibrium with the bathoRT intermediate. PTR/CARS spectra measured at a 210-ns delay contain distinct vibrational features attributable to BSI(RT), which suggest that the all-trans retinal in both BSI(RT) and lumiRT is strongly coupled to part of the retinal binding pocket. With regard to the energy storage/transduction mechanism in RhRT, these results support the hypothesis that during the formation of lumiRT, the majority of the photon energy absorbed by RhRT transfers to the apoprotein opsin.