Excited-state structure and isomerization dynamics of the retinal chromophore in rhodopsin from resonance Raman intensities.

Resonance Raman excitation profiles have been measured for the bovine visual pigment rhodopsin using excitation wavelengths ranging from 457.9 to 647.1 nm. A complete Franck-Condon analysis of the absorption spectrum and resonance Raman excitation profiles has been performed using an excited-state, time-dependent wavepacket propagation technique. This has enabled us to determine the change in geometry upon electronic excitation of rhodopsin's 11-cis-retinal protonated Schiff base chromophore along 25 normal coordinates. Intense low-frequency Raman lines are observed at 98, 135, 249, 336, and 461 cm-1 whose intensities provide quantitative, mode-specific information about the excited-state torsional deformations that lead to isomerization. The dominant contribution to the width of the absorption band in rhodopsin results from Franck-Condon progressions in the 1,549 cm-1 ethylenic normal mode. The lack of vibronic structure in the absorption spectrum is shown to be caused by extensive progressions in low-frequency torsional modes and a large homogeneous linewidth (170 cm-1 half-width) together with thermal population of low-frequency modes and inhomogeneous site distribution effects. The resonance Raman cross-sections of rhodopsin are unusually weak because the excited-state wavepacket moves rapidly (approximately 35 fs) and permanently away from the Franck-Condon geometry along skeletal stretching and torsional coordinates.     

The structure of the retinylidene chromophore in bathorhodopsin.

Resonance Raman data on bathorhodopsin (bovine and squid) at 95,77, and 4 degrees K support a mechanism of excitation proposed by Lewis in which both a protein conformational transition and chromophore structural alteration to a "dicisoid" configuration are required to generate the bathorhodopsin species observed in steady-state photostationary mixtures. However, these results also suggest that the molecular structure with a red-shifted chromophore absorption detected at room temperatures in 1 ps using picosecond absorption spectroscopy may not necessarily have the same chromophore conformation as the steady-state bathorhodopsin species.     

Structure of the retinal chromophore in sensory rhodopsin I from resonance Raman spectroscopy

Sensory rhodopsin I (SR-I) is a retinal-containing pigment which functions as a phototaxis receptor in Halobacterium halobium. We have obtained resonance Raman vibrational spectra of the native membrane-bound form of SR587 and used these data to determine the structure of its retinal prosthetic group. The similar frequencies and intensities of the skeletal fingerprint modes in SR587, bacteriorhodopsin (BR568), and halorhodopsin (HR578) as well as the position of the dideuterio rocking mode when SR-I is regenerated with 12,14-D2 retinal (915 cm-1) demonstrate that the retinal chromophore has an all-trans configuration. The shift of the C = N stretching mode from 1628 cm-1 in H2O to 1620 cm-1 in D2O demonstrates that the chromophore in SR587 is bound to the protein by a protonated Schiff base linkage. The small shift of the 1195 cm-1 C14-C15 stretching mode in D2O establishes that the protonated Schiff base bond has an anti configuration. The low value of the Schiff base stretching frequency together with its small 8 cm-1 shift in D2O indicates that the Schiff base proton is weakly hydrogen bonded to its protein counterion. This suggests that the red shift in the absorption maximum of SR-I (587 nm) compared with HR (578 nm) and BR (568 nm) is due to a reduction of the electrostatic interaction between the protonated Schiff base group and its protein counterion.     

Orientational changes of the absorbing dipole or retinal upon the conversion of rhodopsin to bathorhodopsin, lumirhodopsin, and isorhodopsin.

The orientational change of the absorbing dipole of the retinal chromophore in vertebrate rhodopsin (rhodo) upon photo-excitation to bathorhodopsin (batho), lumirhodopsin (lumi) and isorhodopsin (iso), has been studied by polarized absorption and linear dichroism measurements on magnetically oriented frog rod suspensions that were blocked at liquid nitrogen temperature. Both the azimuthal component delta theta and the polar component delta theta of the total angular change were studied in separate experiments. Delta theta was estimated from polarized absorption measurements on rods oriented transversally with respect to the analyzing beam. The data show unequivocally that upon the rhodo leads to batho transition, the dipole shifts out of the membrane plane by only few degrees; delta theta congruent to -3 degree. This azimuthal shift was nearly exactly reversed upon the batho leads to lumi decay. A very small shift (delta theta less than or equal to 1 degree) toward the membrane plane was observed upon a rhodo leads to iso conversion. The polar component delta theta of the angular shift was estimated by studying the photoreversion of linear dichroism induced by photo-excitation with polarized light in rods oriented parallel to the analyzing beam. Upon the rhodo leads to batho transition, ther was a shift delta theta = 11 +/- 3 degrees. The overall angular shift upon this first photo-exciting step, which corresponded to the isomerisation of retinal, was only delta omega = 11 +/- 3 degrees. This is smaller than what may be expected for a cis-trans isomerization of a retinal molecule with one end fixed, and different from what has been previously estimated by another group. These discrepancies are discussed.     

Complete assignment of the hydrogen out-of-plane wagging vibrations of bathorhodopsin: chromophore structure and energy storage in the primary photoproduct of vision

Resonance Raman vibrational spectra of the retinal chromophore in bathorhodopsin have been obtained after regenerating bovine visual pigments with an extensive series of 13C- and deuterium-labeled retinals. A low-temperature spinning cell technique was used to produce high-quality bathorhodopsin spectra exhibiting resolved hydrogen out-of-plane wagging vibrations at 838, 850, 858, 875, and 921 cm-1. The isotopic shifts and a normal coordinate analysis permit the assignment of these lines to the HC7 = C8H Bg, C14H, C12H, C10H, and C11H hydrogen out-of-plane wagging modes, respectively. The coupling constant between the C11H and C12H wags as well as the C12H wag force constant are unusually low compared to those of retinal model compounds. This quantitatively confirms the lack of coupling between the C11H and C12H wags and the low C12H wag vibrational frequency noted earlier by Eyring et al. [(1982) Biochemistry 21, 384]. The force constants for the C10H and C14H wags are also significantly below the values observed in model compounds. We suggest that the perturbed hydrogen out-of-plane wagging and C-C stretching force constants for the C10-C11 = C12-C13 region of the chromophore in bathorhodopsin result from electrostatic interactions with a charged protein residue. This interaction may also contribute to the 33 kcal/mol energy storage in bathorhodopsin.     

Resonance Raman spectroscopy of chemically modified retinals: Assigning the carbon-methyl vibrations in the resonance Raman spectrum of rhodopsin

To assign the observed vibrationsl modes in the resonance Raman spectrum of the retinylidene chromophore of rhodopsin, we have studied chemically modified retinals. The series of analogs investigated are the n-butyl retinals substituted at C₉ and C₁₃. The results obtained for the 11-cis isomer have clearly assigned the CCH₃ vibrational frequencies observed in the spectrum of the retinylidene chromophore. The data show that the C₍₉₎CH₃ stretching vibration can be assigned to the vibrational mode observed in the 1017 cm^?1 region, and the vibration detected at 997 cm^?1 can be assigned to the C₍₁₃CH₃ vibration. The C₍₅₎CH₃ stretching mode does not contribute to the vibrations observed in this region. The splitting in the C_(n)CH₃ (n = 9, 13) vibration is characteristic of the 11-cis conformation. The results on the modified retinals do not support the hypothesis that the splitting arises from equilibrium mixtures of 11-cis, 12-s-cis and 11-cis, 12-s-trans in solution. Thus, this splitting cannot be used to determine whether the chromophore in rhodopsin is in a 12-s-cis or 12-s-trans conformation. However, our results demonstrate that there are other vibrational modes in the spectra which are sensitive to this conformational equilibrium and we use the presence of a strong ~ 1271 cm^?1 mode in bovine and squid rhodopsin spectra as an indication that the chromophore in these pigments is 11-cis, 12-s-trans.     

Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy

The analysis of the vibrational spectrum of the retinal chromophore in bacteriorhodopsin with isotopic derivatives provides a powerful "structural dictionary" for the translation of vibrational frequencies and intensities into structural information. Of importance for the proton-pumping mechanism is the unambiguous determination of the configuration about the C13=C14 and C=N bonds, and the protonation state of the Schiff base nitrogen. Vibrational studies have shown that in light-adapted BR568 the Schiff base nitrogen is protonated and both the C13=C14 and C=N bonds are in a trans geometry. The formation of K625 involves the photochemical isomerization about only the C13=C14 bond which displaces the Schiff base proton into a different protein environment. Subsequent Schiff base deprotonation produces the M412 intermediate. Thermal reisomerization of the C13=C14 bond and reprotonation of the Schiff base occur in the M412------O640 transition, resetting the proton-pumping mechanism. The vibrational spectra can also be used to examine the conformation about the C--C single bonds. The frequency of the C14--C15 stretching vibration in BR568, K625, L550 and O640 argues that the C14--C15 conformation in these intermediates is s-trans. Conformational distortions of the chromophore have been identified in K625 and O640 through the observation of intense hydrogen out-of-plane wagging vibrations in the Raman spectra (see Fig. 2). These two intermediates are the direct products of chromophore isomerization. Thus it appears that following isomerization in a tight protein binding pocket, the chromophore cannot easily relax to a planar geometry. The analogous observation of intense hydrogen out-of-plane modes in the primary photoproduct in vision (Eyring et al., 1982) suggests that this may be a general phenomenon in protein-bound isomerizations. Future resonance Raman studies should provide even more details on how bacterio-opsin and retinal act in concert to produce an efficient light-energy convertor. Important unresolved questions involve the mechanism by which the protein catalyzes deprotonation of the L550 intermediate and the mechanism of the thermal conversion of M412 back to BR568. Also, it has been shown that under conditions of high ionic strength and/or low light intensity two protons are pumped per photocycle (Kuschmitz & Hess, 1981). How might this be accomplished?(ABSTRACT TRUNCATED AT 400 WORDS)     

Energy storage in the primary photochemical events of rhodopsin and isorhodopsin

The energetics associated with the photoequilibrium (Formula: see text) are measured at 77 K by using pulsed-laser photocalorimetry and a range of excitation wavelengths and relative starting concentrations. Enthalpies for the photochemical transformations R hv----B and I hv----B are measured to be delta HRB = 32.2 +/- 0.9 kcal mol-1 and delta HIB = 27.1 +/- 3.2 kcal mol-1, respectively. Although the value of delta HRB is slightly lower than that reported previously by Cooper of 34.7 +/- 2.2 kcal mol-1 [Cooper, A. (1979) Nature (London) 282, 531-533], the two values are in agreement within experimental error. The energy difference delta HRB - delta HIB = 5.1 +/- 3.3 kcal mol-1 is identical within experimental error with the difference in enthalpies of isorhodopsin and rhodopsin [5.2 +/- 2.3; Cooper, A. (1979) FEBS Lett. 100, 382-384]. We suggest that this result is consistent with the theory that bathorhodopsin is a single, common photochemical intermediate connecting rhodopsin and isorhodopsin.     

Crystal structure of rhodopsin: implications for vision and beyond

A heptahelical transmembrane bundle is a common structural feature of G-protein-coupled receptors (GPCRs) and bacterial retinal-binding proteins, two functionally distinct groups of membrane proteins. Rhodopsin, a photoreceptor protein involved in photopic (rod) vision, is a prototypical GPCR that contains 11-cis-retinal as its intrinsic chromophore ligand. Therefore, uniquely, rhodopsin is a GPCR and also a retinal-binding protein, but is not found in bacteria. Rhodopsin functions as a typical GPCR in processes that are triggered by light and photoisomerization of its ligand. Bacteriorhodopsin is a light-driven proton pump with an all-trans-retinal chromophore that photoisomerizes to 13-cis-retinal. The recent crystal structure determination of bovine rhodopsin revealed a structure that is not similar to previously established bacteriorhodopsin structures. Both groups of proteins have a heptahelical transmembrane bundle structure, but the helices are arranged differently. The activation of rhodopsin involves rapid cis-trans photoisomerization of the chromophore, followed by slower and incompletely defined structural rearrangements. For rhodopsin and related receptors, a common mechanism is predicted for the formation of an active state intermediate that is capable of interacting with G proteins.     

The nature of the primary photochemical events in rhodopsin and isorhodopsin.

The nature of the primary photochemical events in rhodopsin and isorhodopsin is studied by using low temperature actinometry, low temperature absorption spectroscopy, and intermediate neglect of differential overlap including partial single and double configuration interaction (INDO-PSDCI) molecular orbital theory. The principal goal is a better understanding of how the protein binding site influences the energetic, photochemical, and spectroscopic properties of the bound chromophore. Absolute quantum yields for the isorhodopsin (I) to bathorhodopsin (B) phototransformation are assigned at 77 K by using the rhodopsin (R) to bathorhodopsin phototransformation as an internal standard (phi R----B = 0.67). In contrast to rhodopsin photochemistry, isorhodopsin displays a wavelength dependent quantum yield for photochemical generation of bathorhodopsin at 77 K. Measurements at seven wavelengths yielded values ranging from a low of 0.089 +/- 0.021 at 565 nm to a high of 0.168 +/- 0.012 at 440 nm. An analysis of these data based on a variety of kinetic models suggests that the I----B phototransformation encounters a small activation barrier (approximately 0.2 kcal mol-1) associated with the 9-cis----9-trans excited-state torsional-potential surface. The 9-cis retinal chromophore in solution (EPA, 77 K) has the smallest oscillator strength relative to the other isomers: 1.17 (all-trans), 0.98 (9-cis), 1.04 (11-cis), and 1.06 (13-cis). The effect of conformation is quite different for the opsin-bound chromophores. The oscillator strength of the lambda max absorption band of I is observed to be anomalously large (1.11) relative to the lambda max absorption bands of R (0.98) and B (1.07). The wavelength-dependent photoisomerization quantum yields and the anomalous oscillator strength associated with isorhodopsin provide important information on the nature of the opsin binding site. Various models of the binding site were tested by using INDO-PSDCI molecular orbital theory to predict the oscillator strengths of R, B, and I and to calculate the barriers and energy storage associated with the photochemistry of R and I for each model. Our experimental and theoretical investigation leads to the following conclusions: (a) The counterion (abbreviated as CTN) is not intimately associated with the imine proton in R, B, or I. The counterion lies underneath the plane of the chromophore in R and I, and the primary chromophore-counterion electrostatic interactions involve C15-CTN and C13-CTN. These interactions are responsible for the anomalous oscillator strength of I relative to R and B.(ABSTRACT TRUNCATED AT 400 WORDS)     

A study of the Schiff base mode in bovine rhodopsin and bathorhodopsin

We have obtained the resonance Raman spectra of bovine rhodopsin, bathorhodopsin, and isorhodopsin for a series of isotopically labeled retinal chromophores. The specific substitutions are at retinal's protonated Schiff base moiety and include -HC = NH+-, -HC = ND+-, -H13C = NH+-, and -H13C = ND+-. Apart from the doubly labeled retinal, we find that the protonated Schiff base frequency is the same, within experimental error, for both rhodopsin and bathorhodopsin for all the substitutions measured here and elsewhere. We develop a force field that accurately fits the observed ethylenic (C = C) and protonated Schiff base stretching frequencies of rhodopsin and labeled derivatives. Using MINDO/3 quantum mechanical procedures, we investigate the response of this force field, and the ethylenic and Schiff base stretching frequencies, to the placement of charges close to retinal's Schiff base moiety. Specifically, we find that the Schiff base frequency should be measurably affected by a 3.0-4.5-A movement of a negatively charged counterion from the positively charged protonated Schiff base moiety. That there is no experimentally discernible difference in the Schiff base frequency between rhodopsin and bathorhodopsin suggests that models for the efficient conversion of light to chemical energy in the rhodopsin to bathorhodopsin photoconversion based solely on salt bridge separation of the protonated Schiff base and its counterion are probably incorrect. We discuss various alternative models and the role of electrostatics in the rhodopsin to bathorhodopsin primary process.     

Low-frequency vibrations in resonance Raman spectra of horse heart myoglobin. Iron-ligand and iron-nitrogen vibrational modes.

The low-frequency regions (150--700 cm-1) of resonance Raman (RR) spectra of various complexes of oxidized and reduced horse heart myoglobin were examined by use of 441.6-nm excitation. In this frequency range, RR spectra show 10 bands common to all myoglobin derivatives (numbered here for convenience from I to X). Relative intensities of bands IV, V, and X constitute good indicators of the doming state of the heme and, consequently, of the spin state of the iron atom. An additional band is present for several complexes (fluorometmyoglobin, hydroxymetmyoglobin, azidometmyoglobin, and oxymyoglobin). Isotopic substitutions on the exogenous ligands and of the iron atom (56Fe leads to 54Fe) allow us to assign these additional lines to the stretching vibrations of the Fe-sixth ligand bond. Similarly, bands II are assigned to stretching vibrations of the Fe-N-(pyrrole) bonds. An assignment of bands VI to stretching vibrations of the Fe-Nepsilon(proximal histidine) bonds is also proposed. Mechanisms for the resonance enhancement of the main low-frequency bands are discussed on the basis of the excitation profiles and of the dispersion curves for depolarization ratios obtained for fluorometmyoglobin and hydroxymetmyoglobin.     

Assignment of the hydrogen-out-of-plane and -in-plane vibrations of the retinal chromophore in the K intermediate of pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor protein for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all-trans to 13-cis initiates conformational changes of the protein leading to activation of the cognate transducer protein (pHtrII). Elucidation of the initial photoreaction, formation of the K intermediate of ppR, is important for understanding the mechanism of storage of photon energy. We have reported the K minus ppR Fourier transform infrared (FTIR) spectra, including several vibrational bands of the retinal, the protein, and internal water molecules. It is interesting that more vibrational bands were observed in the hydrogen-out-of-plane (HOOP) region than for the light-driven proton pump, bacteriorhodopsin. This result implied that the steric constraints on the retinal chromophore in the binding pocket of ppR are distributed more widely upon formation of the initial intermediate. In this study, we assigned the HOOP and hydrogen-in-plane vibrations by means of low-temperature FTIR spectroscopy applied to ppR reconstituted with retinal deuterated at C7, C8, C10-C12, C14, and C15. As a result, the 966 (+)/971 (-) and 958 (+)/961 (-) cm(-1) bands were assigned to the C7=C8 and C11=C12 Au HOOP modes, respectively, suggesting that the structural changes spread to the middle part of the retinal. The positive bands at 1001, 994, 987, and 979 cm(-1) were assigned to the C15-HOOP vibrations of the K intermediate, whose frequencies are similar to those of the K(L) intermediate of bacteriorhodopsin trapped at 135 K. Another positive band at 864 cm(-1) was assigned to the C14-HOOP vibration. Relatively many positive bands of hydrogen-in-plane vibrations supported the wide distribution of structural changes of the retinal as well. These results imply that the light energy was stored mainly in the distortions around the Schiff base region while some part of the energy was transferred to the distal part of the retinal.     

all-trans-retinoids and dihydroretinoids as probes of the role of chromophore structure in rhodopsin activation

The absorption of a photon of light by rhodopsin results in the cis to trans isomerization of the 11-cis-retinal Schiff base chromophore. In the studies reported here, an attempt is made to determine the mechanism of the energization of rhodopsin as it relates to the chemistry of the isomerization process and the geometrical state of the chromophore. Studies were performed with vitamin A analogues to probe this mechanism. Both 11-cis-7,8-dihydroretinal and 9-cis-7,8-dihydroretinal form bleachable pigments when combined with opsin. Photolysis of these pigments in the presence of G-protein results in the activation of the latter as revealed by its GTPase activity. Phosphodiesterase is also activated when it is included in the incubation. Therefore, the possibility that rhodopsin is energized by mechanisms involving photochemically induced charge transfer from the protonated Schiff base to the beta-ionone ring can be discarded. Further studies were conducted with all-trans-vitamin A derivatives to determine if these compounds can form the GTPase-activating state R*, a situation that is possible, in principle, by microscopic reversibility. Neither all-trans-retinal nor its oxime, when incubated with bovine opsin in the dark, caused activation of the GTPase, requiring at least a 5 kcal/mol energy gap between them. Furthermore, stoichiometric adducts of all-trans-retinoids and opsin were also unable to mediate activation of the GTPase. Since both all-trans-15,16-dihydroretinylopsin and all-trans-retinoylopsin possess an all-trans-retinoid permanently adducted to opsin, it can be concluded that the all-trans-retinoid chromophore-opsin linkage may be necessary but not sufficient to achieve activation of the visual pigment.     

11-cis-retinal protonated Schiff base: influence of the protein environment on the geometry of the rhodopsin chromophore

Density functional theory (DFT) calculations based on the self-consistent-charge tight-binding approximation have been performed to study the influence of the protein pocket on the 3-dimensional structure of the 11-cis-retinal Schiff base (SB) chromophore. Starting with an effectively planar chromophore embedded in a protein pocket consisting of the 27 next-nearest amino acids, the relaxed chromophore geometry resulting from energy optimization and molecular dynamics (MD) simulations has yielded novel insights with respect to the following questions: (i) The conformation of the beta-ionone ring. The protein pocket tolerates both conformations, 6-s-cis and 6-s-trans, with a total energy difference of 0.7 kcal/mol in favor of the former. Of the two possible 6-s-cis conformations, the one with a negative twist angle (optimized value: -35 degrees ) is strongly favored, by 3.6 kcal/mol, relative to the one in which the dihedral is positive. (ii) Out-of-plane twist of the chromophore. The environment induces a nonplanar helical deformation of the chromophore, with the distortions concentrated in the central region of the chromophore, from C10 to C13. The dihedral angle between the planes formed by the bonds from C7 to C10 and from C13 to C15 is 42 degrees. (iii) The absolute configuration of the chromophore. The dihedral angle about the C12-C13 bond is +170 degrees from planar s-cis, which imparts a positive helicity on the chromophore, in agreement with earlier considerations based on theoretical and spectroscopic evidence.     

Formation, conversion, and utilization of isorhodopsin, rhodopsin, and porphyropsin by rod photoreceptors in the Xenopus retina

The visual pigment content of rod photoreceptors in Xenopus larvae was reduced greater than 90% through a combination of vitamin A-deficient diet and constant light. Thereafter, a dose of either all-trans-retinol or 9-cis-retinal was injected intramuscularly, leading to the formation of a rhodopsin (lambdamax 504 nm) or isorhodopsin (lambdamax 487-493 nm) pigment, respectively. Electrophysiological measurements were made of the threshold and spectral sensitivity of the aspartate-isolated PIII (photoreceptoral) component of the electroretinogram. These measures established that either rhodopsin or isorhodopsin subserved visual transduction with the same efficiency as the 519 nm porphyropsin pigment encountered normally. When animals with rhodopsin or isorhodopsin were kept in darkness or placed on a cyclical lighting regimen for 8 days, retinal densitometry showed that either pigment was being converted to porphyropsin; significantly more porphyropsin was formed as a result of cyclical lighting than after complete darkness.     

Cysteine ligand vibrations are responsible for the complex resonance Raman spectrum of azurin

 In the redox center of azurin, the Cu(II) is strongly coordinated to one thiolate S from Cys 112 and two imidazole Ns from His 46 and 117. This site yields a complex resonance Raman (RR) spectrum with >20 vibrational modes between 200 and 1500 cm^–1. We have investigated the effects of ligand-selective isotope replacements on the RR spectrum of Pseudomonas aeruginosa azurin to determine the relative spectral contribution from each of the copper ligands. Growth on ³⁴S-sulfate labels the cysteine ligand and allows the identification of a cluster of bands with Cu–S(Cys) stretching character between 370 and 430 cm^–1 whose frequencies are consistent with the trigonal or distorted tetrahedral coordination in type 1 sites. In type 2 copper-cysteinate sites, the lower ν (Cu–S) frequencies between 260 and 320 cm^–1 are consistent with square-planar coordination. Addition of exogenous ¹⁵N-labeled imidazole or histidine to the His117Gly mutant generates type 1 or type 2 sites, respectively. Because neither the above nor the His46Gly mutant reconstituted with ¹⁵N-imidazole exhibits significant isotope dependence, the histidine ligands can be ruled out as important contributors to the RR spectrum. Instead, a variety of evidence, including extensive isotope shifts upon global substitution with ¹⁵N, suggests that the multiple RR modes of azurin are due principally to vibrations of the cysteine ligand. These are resonance-enhanced through kinematic coupling with the Cu–S stretch in the ground state or through an excited-state A-term mechanism involving a Cu-cysteinate chromophore that extends into the peptide backbone. Received: 29 July 1996 / Accepted: 9 November 1996     

Resonance Raman study of halorhodopsin photocycle kinetics, chromophore structure, and chloride-pumping mechanism.

Kinetic resonance Raman spectra of the HR520, HR640, and HR578 species in the halorhodopsin photocycle are obtained using time delays ranging from 5 microseconds to 10 ms in 0.3 M NO3-, 0.3 M Cl-, and 3 M Cl-. The Raman intensities are converted to absolute concentrations by using a conservation of molecules constraint. The simplest kinetic scheme that satisfactorily models the data is HR578-->HR520 in equilibrium with HR640-->HR578. The rate constant for the HR640-->HR578 transition increases with Cl- concentration, suggesting that Cl- is taken up between HR640 and HR578. The ratio of the forward to the reverse rate constants connecting HR520 and HR640 increases as the inverse of the Cl- concentration, suggesting that Cl- is released during the HR520-->HR640 step. The configuration about the C13 = C14 bond of the retinal chromophore in HR640 is examined by regenerating the protein with [12,14-2H2]retinal. The C12-2H + C14-2H rocking vibration for HR640 is observed at 943 cm-1, demonstrating that the chromophore is 13-cis. The changes in the resonance Raman spectrum of HR640 in response to 2H2O suspension indicates that the Schiff base linkage to the protein is protonated. None of the HR640 fingerprint vibrations shift significantly in 2H2O, suggesting that the Schiff base adopts a C = N anti configuration; this assignment is supported by the frequency of the C15-2H rocking mode (1002 cm-1). The 13-cis structure for the chromophore in HR640 requires that thermal isomerization back to all-trans occurs in the HR640-->HR578 transition. These structural and kinetic results are incorporated into a two-state C-T model for Cl- pumping.