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...

Phytochromes are bimodal photoswitches composed of a photosensor and an output module. Photoactivation of the sensor is initiated by a double bond isomerization of the tetrapyrrole chromophore and eventually leads to protein conformational changes. Recently determined structural models of phytochromes identify differences between the inactive and the signalling state but do not reveal the mechanism of photosensor activation or deactivation. Here, we report a vibrational spectroscopic study on bathy phytochromes that demonstrates that the formation of the photoactivated state and thus (de)activation of the output module is based on proton translocations in the chromophore pocket coupling chromophore and protein structural changes. These proton transfer steps, involving the tetrapyrrole and a nearby histidine, also enable thermal back-isomerization of the chromophore via keto-enol tautomerization to afford the initial dark state. Thus, the same proton re-arrangements inducing the (de)activation of the output module simultaneously initiate the reversal of this process, corresponding to a negative feedback mechanism.

The molecular events during the photocycle of bacteriorhodopsin have been studied by the method of time-resolved and static infrared difference spectroscopy. Characteristic spectral changes involving the C=O stretching vibration of protonated carboxylic groups were detected. To identify the corresponding groups with either glutamic or aspartic acid, BR was selectively labeled with [4-13C]aspartic acid. An incorporation of ca. 70% was obtained. The comparison of the difference spectra in the region of the CO2- stretching vibrations of labeled and unlabeled BR indicates that ionized aspartic acids are influenced during the photocycle, the earliest effect being observed already at the K610 intermediate. Taken together, the results provide evidence that four internal aspartic acids undergo protonation changes and that one glutamic acid, remaining protonated, is disturbed. The results are discussed in relation to the various aspects of the proton pumping mechanism, such as retinal isomerization, charge separation, pK changes, and proton pathway.

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The photoreaction of opsin regenerated with 9-demethylretinal has been investigated by UV-vis spectroscopy, flash photolysis experiments, and Fourier transform infrared difference spectroscopy. In addition, the capability of the illuminated pigment to activate the retinal G-protein has been tested. The photoproduct, which can be stabilized at 77 K, resembles more the lumirhodopsin species, and only minor further changes occur upon warming the sample to 170 K (stabilizing lumirhodopsin). UV-vis spectroscopy reveals no further changes at 240 K (stabilizing metarhodopsin I), but infrared difference spectroscopy shows that the protein as well as the chromophore undergoes further molecular changes which are, however, different from those observed for unmodified metarhodopsin I. UV-vis spectroscopy, flash photolysis experiments, and infrared difference spectroscopy demonstrate that an intermediate different from metarhodopsin II is produced at room temperature, of which the Schiff base is still protonated. The illuminated pigment was able to activate G-protein, as assayed by monitoring the exchange of GDP for GTP gamma S in purified G-protein, only to a very limited extent (approximately 8% as compared to rhodopsin). The results are interpreted in terms of a specific steric interaction of the 9-methyl group of the retinal in rhodopsin with the protein, which is required to initiate the molecular changes necessary for G-protein activation. The residual activation suggests a conformer of the photolyzed pigment which mimics metarhodopsin II to a very limited extent.