Ultrafast infrared spectroscopy of bacteriorhodopsin.

Picosecond infrared spectroscopy is developed and used for the first time to study the dynamics of photoexcited bacteriorhodopsin (BR). Both spectral and time-resolved data are obtained. The results open an entirely new approach to investigations of the BR photocycle. The infrared difference spectrum (K minus BR570) recorded at ambient temperature between 1,560 and 1,700 cm-1 is not identical with the spectrum reported for a frozen sample. Three bands of the K state at 1,622, 1,610, and 1,580 cm-1 and the bleaching at 1,637 cm-1 (C = NH stretch) are seen. These new spectral lines appear in less than 10 ps.     

Picosecond time-resolved absorption and fluorescence dynamics in the artificial bacteriorhodopsin pigment BR6.11.

The picosecond molecular dynamics in an artificial bacteriorhodopsin (BR) pigment containing a structurally modified all-trans retinal chromphore with a six-membered ring bridging the C11=C12-C13 positions (BR6.11) are measured by picosecond transient absorption and picosecond time-resolved fluorescence spectroscopy. Time-dependent intensity and spectral changes in absorption in the 570-650-nm region are monitored for delays as long as 5 ns after the 7-ps, 573-nm excitation of BR6.11. Two intermediates, J6.11 and K6.11/1, both with enhanced absorption to the red (> 600 nm) of the BR6.11 spectrum are observed within approximately 50 ps. The J6.11 intermediate decays with a time constant of 12 +/- 3 ps to form K6.11/1. The K6.11/1 intermediate decays with an approximately 100-ps time constant to form a third intermediate, K6.11/2, which is observed through diminished 650-nm absorption (relative to that of K6.11/1). No other transient absorption changes are found during the remainder of the initial 5-ns period of the BR6.11 photoreaction. Fluorescence in the 650-900-nm region is observed from BR6.11, K6.11/1, and K6.11/2, but no emission assignable to J6.11 is found. The BR6.11 fluroescence spectrum has a approximately 725-nm maximum which is blue-shifted by approximately 15 nm relative to that of native BR-570 and is 4.2 +/- 1.5 times larger in intensity (same sample optical density). No differences in the profile of the fluorescence spectra of BR6.11 and the intermediates K6.11/1 and K6.11/2 are observed. Following ground-state depletion of the BR6.11 population, the time-resolved fluroescence intensity monitored at 725 nm increases with two time constants, 12 +/- 3 and approximately 100 ps, both of which correlate well with changes in the picosecond transient absorption data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Asp-96----Asn, Asp-85----Asn, and Arg-82----Gln single-site substitutions on the photocycle of bacteriorhodopsin

Bacteriorhodopsin (BR) with the single-site substitutions Arg-82----Gln (R82Q), Asp-85----Asn (D85N), and Asp-96----Asn (D96N) is studied with time-resolved absorption spectroscopy in the time regime from nanoseconds to seconds. Time-resolved spectra are analyzed globally by using multiexponential fitting of the data at multiple wavelengths and times. The photocycle kinetics for BR purified from each mutant are determined for micellar solutions in two detergents, nonyl glucoside and CHAPSO, and are compared to results from studies on delipidated BR (d-BR) in the same detergents. D85N has a red-shifted ground-state absorption spectrum, and the formation of an M intermediate is not observed. R82Q undergoes a pH-dependent transition between a purple and a blue form with different pKa values in the two detergents. The blue form has a photocycle resembling that for D85N, while the purple form of R82Q forms an M intermediate that decays more rapidly than in d-BR. The purple form of R82Q does not light-adapt to the same extent as d-BR, and the spectral changes in the photocycle suggest that the light-adapted purple form of R82Q contains all-trans- and 13-cis-retinal in approximately equal proportions. These results are consistent with the suggestions of others for the roles of Arg-82 and Asp-85 in the photocycle of BR, but results for D96N suggest a more complex role for Asp-96 than previously suggested. In nonyl glucoside, the apparent decay of the M-intermediate is slower in D96N than in d-BR, and the M decay shows biphasic kinetics. However, the role of Asp-96 is not limited to the later steps of the photocycle. In D96N, the decay of the KL intermediate is accelerated, and the rise of the M intermediate has an additional slow phase not observed in the kinetics of d-BR. The results suggest that Asp-96 may play a role in regulating the structure of BR and how it changes during the photocycle.     

Absorption spectra of intermediates of bacteriorhodopsin measured by laser photolysis at room temperatures

Picosecond laser spectroscopic analysis was applied to determine how many intermediates existed in the primary photochemical process of trans-bacteriorhodopsin (light-adapted bacteriorhodopsin) at room temperature (18°C) and to calculate their absorption spectra. Irradiation of bacteriorhodopsin with a laser pulse (wavelength, 532 nm; pulse width, 25 ps) yielded the K intermediate (K) which was produced through a precursor, having an absorption maximum (λ_max) longer than that of K. K was stable during a picosecond time range (50–900 ps). The λ_max was located at 610 nm and the extinction coefficient (?_max) was 0.92-times that of bacteriorhodopsin. The same K intermediate was produced from bacteriorhodopsin even when it was excited with a high-energy pulse by which a saturation effect was induced. A transient difference spectrum measured at 150 ns after the excitation of bacteriorhodopsin was different in shape from that of the K intermediate, suggesting that an intermediate was formed by thermal decay of K. This intermediate, tentatively called the KL intermediate (KL), had a λ_max at 596 nm and an ?_max 0.80-times that of bacteriorhodopsin. KL decayed to the L intermediate (L) with a time constant of 2.2 μs. L has a λ_max at 543 nm and an ?_max 0.66-times that of bacteriorhodopsin.     

Polarization of the ion atmosphere of a charged cylinder

The dipole moment is calculated for an electric-field-induced polarization of a Debye-Hückel ion atmosphere surrounding a charged rod. If L is the length of a thin rod. Q is its linear charge density, Z is charge of the salt ion in solution, and k is the Debye-Hückel shielding parameter, then for KL less, similar 10, the calculated polarizability is proportional to Z(2)Q(2)L(1.8)/K(1.2). Comparison with experimental data for DNA shows that the ion atmosphere dipole is of the correct magnitude and is consistent with observed variations with Z, Q, L and k.     

Chromophore-protein-water interactions in the L intermediate of bacteriorhodopsin: FTIR study of the photoreaction of L at 80 K.

Using FTIR spectroscopy, perturbations of several residues and internal water molecules have been detected when light transforms all-trans bacteriorhodopsin (BR) to its L intermediate having a 13-cis chromophore. Illumination of L at 80 K results in an intermediate L' absorbing around 550 nm. L' thermally converts to the original BR only at >130 K. In this study, we used the light-induced transformation of L to L' at 80 K to identify some amino acid residues and water molecules that closely interact with the chromophore and distinguish them from those residues not affected by the photoreaction. The L minus L' FTIR difference spectrum shows that the chromophore in L' is in the all-trans configuration. The perturbed states of Asp96 and Val49 and of the environment along the aliphatic part of the retinal and Lys216 seen in L are not affected by the L --> L' photoreaction. On the other hand, the environments of the Schiff base of the chromophore, of Asp115, and of water molecules close to Asp85 returned in L' to their state in which they originally had existed in BR. The water molecules that are affected by the mutations of Thr46 and Asp96 also change to a different state in the L --> L' transition, as indicated by transformation of a water O-H vibrational band at 3497 cm-1 in L into an intense peak at 3549 cm-1 in L'. Notably, this change of water bands in the L --> L' transition at 80 K is entirely different from the changes observed in the BR --> K photoreaction at the same temperature, which does not show such intense bands. These results suggest that these water molecules move closer to the Schiff base as a hydrogen bonding cluster in L and L', presumably to stabilize its protonated state during the BR to L transition. They may contribute to the structural constraints that prevent L from returning to the initial BR upon illumination at 80 K.     

Structural changes of water in the Schiff base region of bacteriorhodopsin: proposal of a hydration switch model

In a light-driven proton-pump protein, bacteriorhodopsin (BR), three water molecules participate in a pentagonal cluster that stabilizes an electric quadrupole buried inside the protein. In low-temperature Fourier transform infrared (FTIR) K minus BR spectra, the frequencies of water bands suggest extremely strong hydrogen bonding conditions in BR. The three observed water O-D stretches, at 2323, 2292, and 2171 cm(-1), are probably associated with water that interacts with the negative charges in the Schiff base region. Retinal isomerization weakens these hydrogen bonds in the K intermediate, but not in the later intermediates such as L, M, and N. In these states, spectral changes of water bands appeared only in the >2500 cm(-1) region, which correspond to weak hydrogen bonds. This observation suggests that after the K state the water molecules in the Schiff base region find a hydrogen bonding acceptor. We propose here a model for the mechanism of proton transfer from the Schiff base to Asp85. In the "hydration switch model", hydration of a water molecule is switched in the M intermediate from Asp85 to Asp212. This will have increased the pK(a) of the proton acceptor, and the proton transfer is from the Schiff base to Asp85. The present results also suggest that the deprotonated Asp96 in the N intermediate is stabilized in a manner different from that of Asp85 in BR.     

Proton transfers in the photochemical reaction cycle of proteorhodopsin

The spectral and photochemical properties of proteorhodopsin (PR) were determined to compare its proton transport steps to those of bacteriorhodopsin (BR). Static and time-resolved measurements on wild-type PR and several mutants were done in the visible and infrared (FTIR and FT-Raman). Assignment of the observed C=O stretch bands indicated that Asp-97 and Glu-108 serve as the proton acceptor and donor, respectively, to the retinal Schiff base, as do the residues at corresponding positions in BR, but there are numerous spectral and kinetic differences between the two proteins. There is no detectable dark-adaptation in PR, and the chromophore contains nearly entirely all-trans retinal. Because the pK(a) of Asp-97 is relatively high (7.1), the proton-transporting photocycle is produced only at alkaline pH. It contains at least seven transient states with decay times in the range from 10 micros to 200 ms, but the analysis reveals only three distinct spectral forms. The first is a red-shifted K-like state. Proton release does not occur during the very slow (several milliseconds) rise of the second, M-like, intermediate, consistent with lack of the residues facilitating extracellular proton release in BR. Proton uptake from the bulk, presumably on the cytoplasmic side, takes place prior to release (tau approximately 2 ms), and coincident with reprotonation of the retinal Schiff base. The intermediate produced by this process contains 13-cis retinal as does the N state of BR, but its absorption maximum is red-shifted relative to PR (like the O state of BR). The decay of this N-like state is coupled to reisomerization of the retinal to all-trans, and produces a state that is O-like in its C-C stretch bands, but has an absorption maximum apparently close to that of unphotolyzed PR.     

Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin.

The time course of structural changes accompanying the transition from the M412 intermediate to the BR568 ground state in the photocycle of bacteriorhodopsin (BR) from Halobacterium halobium was studied at room temperature with a time resolution of 15 ms using synchrotron radiation X-ray diffraction. The M412 decay rate was slowed down by employing mutated BR Asp96Asn in purple membranes at two different pH-values. The observed light-induced intensity changes of in-plane X-ray reflections were fully reversible. For the mutated BR at neutral pH the kinetics of the structural alterations (tau 1/2 = 125 ms) were very similar to those of the optical changes characterizing the M412 decay, whereas at pH 9.6 the structural relaxation (tau 1/2 = 3 s) slightly lagged behind the absorbance changes at 410 nm. The overall X-ray intensity change between the M412 intermediate and the ground state was about 9% for the different samples investigated and is associated with electron density changes close to helix G, B and E. Similar changes (tau 1/2 = 1.3-3.6 s), which also confirm earlier neutron scattering results on the BR568 and M412 intermediates trapped at -180 degrees C, were observed with wild type BR retarded by 2 M guanidine hydrochloride (pH 9.4). The results unequivocally prove that the tertiary structure of BR changes during the photocycle.     

Interaction of Asn105 with the retinal chromophore during photoisomerization of pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR has a blue-shifted absorption spectrum with a spectral shoulder, which is highly unique for the archaeal rhodopsin family. The primary reaction of ppR is a cis-trans photoisomerization of the retinal chromophore to form the K intermediate, like the well-studied proton pump bacteriorhodopsin (BR). Recent comparative FTIR spectroscopy of the K states in ppR and BR revealed that more extended structural changes take place in ppR than in BR with respect to chromophore distortion and protein structural changes [Kandori, H., Shimono, K., Sudo, Y., Iwamoto, M., Shichida, Y., and Kamo, N. (2001) Biochemistry 40, 9238-9246]. FTIR spectroscopy of the N105D mutant protein reported here assigns the vibrational bands at 1704 and 1700 cm(-1) as C=O stretches of Asn105 in ppR and ppR(K), respectively. A comparative investigation between ppR and BR further reveals that the structure at position 105 in ppR is similar to that of the corresponding position (Asp115) in BR; this observation is supported by the recent X-ray crystallographic structures of ppR [Luecke, H., Schobert, B., Lanyi, J. K., Spudich, E. N., and Spudich, J. L. (2001) Science 293, 1499-1503; Royant, A., Nollert, P., Edman, K., Neutze, R., Landau, E. M., Pebay-Peyroulla, E., and Navarro, J. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 10131-10136]. Nevertheless, structural changes upon photoisomerization at position 105 in ppR are greater than those at position 115 in BR. As a consequence of a unique chromophore-protein interaction in ppR, extended protein structural changes accompanying retinal photoisomerization occur, and these include Asn105 which is approximately 7 A from the retinal chromophore.     

Picosecond time-resolved fluorescence spectroscopy of K-590 in the bacteriorhodopsin photocycle.

The fluorescence spectrum of a distinct isometric and conformational intermediate formed on the 10(-11) s time scale during the bacteriorhodopsin (BR) photocycle is observed at room temperature using a two laser, pump-probe technique with picosecond time resolution. The BR photocycle is initiated by pulsed (8 ps) excitation at 565 nm, whereas the fluorescence is generated by 4-ps laser pulses at 590 nm. The unstructured fluorescence extends from 650 to 880 nm and appears in the same general spectral region as the fluorescence spectrum assigned to BR-570. The transient fluorescence spectrum can be distinguished from that assigned to BR-570 by a larger emission quantum yield (approximately twice that of BR-570) and by a maximum intensity near 731 nm (shifted 17 nm to higher energy from the maximum of the BR-570 fluorescence spectrum). The fluorescence spectrum of BR-570 only is measured with low energy, picosecond pulsed excitation at 590 nm and is in good agreement with recent data in the literature. The assignment of the transient fluorescence spectrum to the K-590 intermediate is based on its appearance at time delays longer than 40 ps. The K-590 fluorescence spectrum remains unchanged over the entire 40-100-ps interval. The relevance of these fluorescence data with respect to the molecular mechanism used to model the primary processes in the BR photocycle also is discussed.     

Nanosecond time-resolved infrared spectroscopy distinguishes two K species in the bacteriorhodopsin photocycle.

The photochemical reaction process of bacteriorhodopsin in the nanosecond time range (-120-860 ns) was measured in the 1400-900 cm-1 region with an improved time resolved dispersive-type infrared spectrometer. The system is equipped with a newly developed detection unit whose instrumental response to a 5-ns laser pulse has a full width of the half-maximum of 60 ns. It provides highly accurate data that enabled us to extract a kinetic process one order of magnitude faster than the instrumental response. The spectral changes in the 1400-900 cm-1 region were analyzed by singular value decomposition and resolved into three components. These components were separated by fitting with 10- and 1000-ns exponential functions and a step function, which were convoluted with the instrumental response function. The components with decay time constants of 10 and 1000 ns are named K and KL, respectively, on the basis of previous visible spectroscopy. The spectral shapes of K and KL are distinguishable by their hydrogen-out-of-plane (HOOP) modes, at 958 and 984 cm-1, respectively. The former corresponds to the K intermediate recorded at 77 K and the latter to a K-like photoproduct at 135 K. On the basis of published data, these bands are assigned to the 15-HOOP mode, indicating that the K and KL differ in a twist around the C14-C15 bond.     

FTIR study of the L intermediate of Anabaena sensory rhodopsin: structural changes in the cytoplasmic region

Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria. The gene encoding ASR forms a single operon with ASRT (ASR transducer) that is a 14 kDa soluble protein, suggesting that ASR functions as a photochromic sensor by activating the soluble transducer. One of the characteristics of ASR is that the formation of the M intermediate accompanies a proton transfer from the Schiff base to Asp217 in the cytoplasmic side [Shi, L., Yoon, S. R., Bezerra, A. G., Jr., Jung, K. H., and Brown, L. S. (2006) J. Mol. Biol. 358, 686-700], in remarkable contrast to other archaeal-type rhodopsins such as a light-driven proton-pump, bacteriorhodopsin (BR). In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the all- trans form of ASR at 170 K, and compared the structural changes in the L intermediate with those of BR. The ASR L minus ASR difference spectra were essentially similar to those for BR, suggesting common structures for the L state in ASR and BR. On the other hand, unique CO stretching bands of a protonated carboxylic acid were observed at 1722 (+) and 1703 (-) cm (-1) at pH 5 and 7, and assigned to Glu36 by use of mutants. Glu36 is located at the cytoplasmic side, and the distance from the Schiff base is about 20 A. This result shows the structural changes at the cytoplasmic surface in ASR L. pH-dependent frequency change was also observed for a water stretching vibration, suggesting that the water molecule is involved in a hydrogen-bonding network with Glu36 and Asp217. Unique hydrogen-bonding network in the cytoplasmic domain of ASR will be discussed.     

Temperature and anation studies of the type 2 site in Rhus vernicifera laccase

Temperature-dependent structural changes involving the type 2 site in laccase are probed by EPR studies of a derivative of laccase in which the type 1 Cu has been replaced by Hg(II) [Morie-Bebel, M. M., Morris, M. C., Menzie, J. L., & McMillin, D. R. (1984) J. Am. Chem. Soc. 106, 3677-3678]. At the temperature extremes (123 and 299 K), single well-defined species are present, but at intermediate temperatures (between 213 and 253 K), the presence of multiple structures is indicated. For the first time, the room temperature EPR spectrum of the type 2 copper has been resolved. Azide binding and fluoride binding have also been studied as a function of temperature. The results suggest that each anion preferentially interacts with the type 3 site in fluid solution and that these adducts can be trapped by rapidly cooling the sample to 123 K. Annealing the adducts at 253 K permits rearrangement and binding at an equatorial position of the type 2 Cu. This pathway to anation at the type 2 site contrasts sharply with previous studies which required a large excess of anions, and it reveals important insight into the flexibility of the type 2/type 3 cluster in laccase.     

Vibrational frequency and dipolar orientation of the protonated Schiff base in bacteriorhodopsin before and after photoisomerization

Light-driven proton transport in bacteriorhodopsin (BR) is initiated by photoisomerization of the retinylidene chromophore, which perturbs the hydrogen bonding network in the Schiff base region of the active site. This study aimed to identify the frequency and dipolar orientation of the N-D stretching vibrations of the Schiff base before and after photoisomerization, by means of low-temperature polarized FTIR spectroscopy of [zeta-(15)N]lysine-labeled BR in D(2)O. (15)N-shifted modes were found at 2123 and 2173 cm(-1) for BR, and at 2468 and 2495 cm(-1) for the K intermediate. The corresponding N-H stretches are at approximately 2800 cm(-1) for BR and 3350-3310 cm(-1) for the K intermediate. The shift to a 350 cm(-1) higher frequency upon photoisomerization is consistent with loss of the hydrogen bond of the Schiff base. The N-D stretch frequencies of the Schiff base in BR and the K intermediate are close to the O-D stretch frequencies of strongly hydrogen bonded water and Thr89, respectively. The angles of the dipole moments of the N-D stretches to the membrane normal were determined to be 60-65 degrees for BR and approximately 90 degrees for the K intermediate. In the case of BR, the stretch orientation is expected to deviate from the N-D bond orientation due to vibrational mixing in the hydrogen bonding network. In contrast, the data for the K intermediate suggest that the N-D group is not hydrogen bonded and orients along the membrane.     

EPR and low temperature spectrophotometric study of the phototransformation products of bacteriorhodopsin]

It is shown that BR and intermediate products of its phototransformation P600, P550 and P415 (the maximum at -196 degrees C at 419 nm) are not paramagnetic. Illumination of samples containing P415 (P419) at -- 196 degrees C with light in the region of 360-480 nm results in the formation of paramagnetic centres with a sunglet spectrum deltaH=18 Oe and g=2.002 (R1). In parallel formation of a new photoproduct P421 in the absorption spectrum is observed. During subsequent heating at -140 degrees C formation of an asymmetric signal with deltaH=45 Oe and g=2.006 and g=2.03 was observed. In the absorption spectra a dark transition. P421-P565 was observed under the same conditions. P565 differs from initial BR P570 as to its photochemical properties. R1 is identified as retinal radical, R2 as a peroxide radical of the BR-complex lipids. Paramagnetic, spectral, and photochemical properties of some products of BR transformation are compared. A scheme of oxidative-phosphorylation processes with participation of Mn ions in BR phototransformation.     

Volume and enthalpy changes in the early steps of bacteriorhodopsin photocycle studied by time-resolved photoacoustics.

We have studied the photoinduced volume changes, energetics, and kinetics in the early steps of the bacteriorhodopsin (BR) photocycle with pulsed, time-resolved photoacoustics. Our data show that there are two volume changes. The fast volume change ( < or = 200 ns) is an expansion (2.5 +/- 0.3 A3/molecule) and is observed exclusively in the purple membrane (PM), vanishing in the 3-[(3-cholamidopropyl)-dimethylammonio] -1-propane-sulfonate-sulfonate-solubilized BR sample; the slow change (approximately 1 micros) is a volume contraction (-3.7 +/- 0.3 A3/molecule). The fast expansion is assigned to the restructuring of the aggregated BR in the PM, and the 1-micros contraction to the change in hydrogen bonding of water at Asp 212 (Kandori et al. 1995. J. Am. Chem. Soc. 117:2118-2119). The formation of the K intermediate releases most of the absorbed energy as heat, with delta Hk = -36 +/- 8 kJ/mol. The activation energy of the K --> L step is 49 +/- 6 kJ/mol, but the enthalpy change is small, -4 +/- 10 kJ/mol. On the time scale we studied, the primary photochemical kinetics, enthalpy, and volume changes are not affected by substituting the solvent D2O for H2O. Comparing data on monomeric and aggregated BR, we conclude that the functional unit for the photocycle is the BR monomer, because both the kinetics (rate constant and activation energy) and the enthalpy changes are independent of its aggregation state.     

Structures of aspartic acid-96 in the L and N intermediates of bacteriorhodopsin: analysis by Fourier transform infrared spectroscopy.

The light-induced difference Fourier transform infrared spectrum between the L or N intermediate minus light-adapted bacteriorhodopsin (BR) was measured in order to examine the protonated states and the changes in the interactions of carboxylic acids of Asp-96 and Asp-115 in these intermediates. Vibrational bands due to the protonated and unprotonated carboxylic acid were identified by isotope shift and band depletion upon substitution of Asp-96 or -115 by asparagine. While the signal due to the deprotonation of Asp-96 was clearly observed in the N intermediate, this residue remained protonated in L. Asp-115 was partially deprotonated in L. The C = O stretching vibration of protonated Asp-96 of L showed almost no shift upon 2H2O substitution, in contrast to the corresponding band of Asp-96 or Asp-115 of BR, which shifted by 9-12 cm-1 under the same conditions. In the model system of acetic acid in organic solvents, such an absence of the shift of the C = O stretching vibration of the protonated carboxylic acid upon 2H2O substitution was seen only when the O-H of acetic acid is hydrogen-bonded. The non-hydrogen-bonded monomer showed the 2H2O-dependent shift. Thus, the O-H bond of Asp-96 enters into hydrogen bonding upon conversion of BR to L. Its increased hydrogen bonding in L is consistent with the observed downshift of the O-H stretching vibration of the carboxylic acid of Asp-96.     

Tyrosine protonation changes in bacteriorhodopsin. A Fourier transform infrared study of BR548 and its primary photoproduct

The structural alterations which occur in bacteriorhodopsin (bR) during dark adaptation (BR570----BR548) and the primary phototransition of the dark photocycle (BR548----KD610) have been investigated by Fourier transform infrared and UV difference spectroscopy. Possible contributions of tyrosine to the Fourier transform infrared difference spectra of these transitions were assigned by incorporating ring per-deuterated tyrosine into bR. Based on these data and UV difference measurements, we conclude that a stable tyrosinate exists in BR570 at physiological temperature and that it protonates during formation of BR548. A tyrosinate protonation has also been observed at low temperature during the primary phototransition of BR570 to the red-shifted photoproduct K630 (1). However, we now find that no tyrosine protonation change occurs during the primary phototransition of BR548 to the red-shifted intermediate KD610. Through analysis of bR containing isotopically labeled retinals, it was also determined that the chromophore of KD610 exits in a 13-trans, 15-cis configuration. On the basis of this evidence and previous studies on the structure of the chromophore in BR570, BR548, and K630, it appears that only the 13-trans,15-trans configuration of the protonated chromophore leads to a stable tyrosinate group. It is proposed that a tyrosinate residue is stabilized due to its interaction with the Schiff base positive charge in the BR570 chromophore. Isomerization of the chromophore about either the C13 = C14 or C = N bond disrupts this interaction causing a protonation of the tyrosinate.