Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin.

Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with hydrochloric acid showed the transition to a form absorbing at 605 nm (bR605 acid) with pK = 2.9 and to a second form absorbing at 565 nm (bR565 acid) with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a third isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, bR605 acid, and bR565 acid all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-microseconds time resolution) of bR605 acid and bR565 acid showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for bR565 acid. Chromophore extraction from membrane suspensions yielded all-trans-retinal for bR565 acid and a mixture of 13-cis and trans isomers for bR605 acid.     

Effect of lipid-protein interaction on the color of bacteriorhodopsin

Detergent solubilization and subsequent delipidation of bacteriorhodopsin (bR) results in the formation of a new species absorbing maximally at 480 nm (bR480). Upon lowering the pH, its absorption shifts to 540 nm (bR540). The pK of this equilibrium is 2.6, with the higher pH favoring bR480 (Baribeau, J. and Boucher, F. (1987) Biochim. Biophysica Acta, 890, 275-278). Resonance Raman spectroscopy shows that bR480, like the native bR, contains a protonated Schiff base (PSB) linkage between the chromophore and the protein. However, the Schiff base vibrational frequency in bR480, and its shift upon deuteration, are quite different from these in the native bR, suggesting changes in the Schiff base environment upon delipidation. Infrared absorption and circular-dichroism (CD) spectral studies do not show any net change in the protein secondary structure upon formation of bR480. It is shown that deprotonation of the Schiff base is not the only mechanism of producing hypsochromic shift in the absorption maximum of bR-derived pigments, subtle changes in the protein tertiary structure, affecting the Schiff base environment of the chromophore, may play an equally significant role in the color regulation of bR-derived pigments.     

Solid state NMR study of [epsilon-13C]Lys-bacteriorhodopsin: Schiff base photoisomerization.

Previous solid state 13C-NMR studies of bacteriorhodopsin (bR) have inferred the C = N configuration of the retinal-lysine Schiff base linkage from the [14-13C]retinal chemical shift (1-3). Here we verify the interpretation of the [14-13C]-retinal data using the [epsilon-13C]lysine 216 resonance. The epsilon-Lys-216 chemical shifts in bR555 (48 ppm) and bR568 (53 ppm) are consistent with a C = N isomerization from syn in bR555 to anti in bR568. The M photointermediate was trapped at pH 10.0 and low temperatures by illumination of samples containing either 0.5 M guanidine-HCl or 0.1 M NaCl. In both preparations, the [epsilon-13C]Lys-216 resonance of M is 6 ppm downfield from that of bR568. This shift is attributed to deprotonation of the Schiff base nitrogen and is consistent with the idea that the M intermediate contains a C = N anti chromophore. M is the only intermediate trapped in the presence of 0.5 M guanidine-HCl, whereas a second species, X, is trapped in the presence of 0.1 M NaCl. The [epsilon-13C]Lys-216 resonance of X is coincident with the signal for bR568, indicating that X is either C = N anti and protonated or C = N syn and deprotonated.     

Structure of the retinal chromophore in the hR578 form of halorhodopsin

Halorhodopsin is a retinal-containing pigment that is thought to function as a light-driven chloride ion pump in the cell membrane of Halobacterium halobium. To address the role of the retinal chromophore in chloride ion transport, resonance Raman spectra have been obtained of the hR578 form of chromatographically purified halorhodopsin (hR). The close similarity of the frequencies and intensities of the hR578 Raman bands with those of light-adapted bacteriorhodopsin (bR568) shows that the chromophore in hR578 has an all-trans configuration and that the protein environment around the chromophore in these two pigments is very similar. In addition, hR578 exhibits a Raman line at 1633 cm-1 which is assigned as the stretching vibration of a protonated Schiff base linkage to the protein based on its shift to 1627 cm-1 in D2O. The reduced frequency of the Schiff base stretching vibration compared with bR568 (1640 cm-1) is shown to result from a reduction of its coupling with the NH in-plane rock. This may be due to a reduction in hydrogen-bonding between the Schiff base proton and an electronegative counterion in halorhodopsin.     

Structure and protein environment of the retinal chromophore in light- and dark-adapted bacteriorhodopsin studied by solid-state NMR

Our previous solid-state 13C NMR studies on bR have been directed at characterizing the structure and protein environment of the retinal chromophore in bR568 and bR548, the two components of the dark-adapted protein. In this paper, we extend these studies by presenting solid-state NMR spectra of light-adapted bR (bR568) and examining in more detail the chemical shift anisotropy of the retinal resonances near the ionone ring and Schiff base. Magic angle spinning (MAS) 13C NMR spectra were obtained of bR568, regenerated with retinal specifically 13C labeled at positions 12-15, which allowed assignment of the resonances observed in the dark-adapted bR spectrum. Of particular interest are the assignments of the 13C-13 and 13C-15 resonances. The 13C-15 chemical resonance for bR568 (160.0 ppm) is upfield of the 13C-15 resonance for bR548 (163.3 ppm). This difference is attributed to a weaker interaction between the Schiff base and its associated counterion in bR568. The 13C-13 chemical shift for bR568 (164.8 ppm) is close to that of the all-trans-retinal protonated Schiff base (PSB) model compound (approximately 162 ppm), while the 13C-13 resonance for bR548 (168.7 ppm) is approximately 7 ppm downfield of that of the 13-cis PSB model compound. The difference in the 13C-13 chemical shift between bR568 and bR548 is opposite that expected from the corresponding 15N chemical shifts of the Schiff base nitrogen and may be due to conformational distortion of the chromophore in the C13 = C14-C15 bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photovoltage kinetics of the acid-blue and acid-purple forms of bacteriorhodopsin: evidence for no net charge transfer.

Time-resolved photovoltage measurements were performed with the acid-blue (bR605A) and acid-purple (bR565A) forms of bacteriorhodopsin (bR) in the time range from 25 ns to 100 s. The bR605A and bR565A pigments were formed by titration with H2SO4 in the absence and presence of 150 mM KCI, respectively. Qualitatively the kinetics of the charge displacement in these two states are similar and consist of two fast phases in one direction (100 ns bandwidth limited and approximately 1 microsecond) followed by a decay in the opposite direction via one component for bR605A (4.4 +/- 0.6 ms) or two components for bR565A (33 +/- 8 microseconds and 3.6 +/- 0.5 ms). The transient photovoltage signal returns exactly to the initial value after several milliseconds, well before the passive discharge of the electrical measuring system at 2 s. We conclude that no net charge transfer occurs in either bR605A or bR565A. The direction of the fast components is opposite that of net proton translocation in bR at pH 7. So, if the charge that moves back and forth is due to a proton, it moves first in the direction of the cytoplasmic side of the membrane (< 1 microsecond) and returns to its initial position via the 4.4 ms (bR605A) or the 33 microseconds and 3.6 ms (bR565A) decay components. The amplitude of the charge motion in both low pH forms is too large to be due to isomerization alone and is comparable to one of the major components in bR at pH 7.2     

Protein-chromophore interactions in bacteriorhodopsin: the effects of a change in surface potential.

The chromophore retinal is bound to bacteriorhodopsin via a protonated Schiff base linkage. The retinal binding site is reported to be buried in the transmembrane portion of the protein, distant from the membrane surfaces. When bound to bacteriorhodopsin, the absorption maximum of retinal is red-shifted from 366 nm to 568 nm producing a purple color. This color persists across a wide pH range. However, when the pH is raised above 12.0, the membranes become pink in color, while at pH values of 3.0 or below, a blue color is produced. The blue color can also be obtained by removing the divalent cations bound to the surface of the protein. In this study, bacteriorhodopsin was examined by circular dichroism and absorption spectroscopy to determine if protein conformational changes were associated with the color shifts. It was found that although the retinal chromophore can be completely removed by bleaching with hydroxylamine with no significant influence on the secondary structure of the protein, a change in the surface charge of bacteriorhodopsin results in measurable conformational change in the protein, which apparently affects the nature of the retinal binding site.     

Time-resolved absorbance changes induced by fast acidification of bacteriorhodopsin in vesicle systems.

The direction of the accessibility to protons of the binding site in bacteriorhodopsin is of primary importance in elucidating the proton-pump mechanism. The problem is approached via the pH-dependent equilibrium bR560 in equilibrium bR605 in vesicles with preferentially oriented purple membranes. Fast acidification (stopped-flow) experiments with inside-out, monomeric, bR vesicles were carried out with and without a buffer enclosed in the vesicle interior. The results, showing a buffer-induced delay in the formation of bR605, indicate that the binding site is accessible to protons from the inside of the vesicles. We arrive at this conclusion also by working with inside-out trimeric vesicles in the presence and in the absence of H+ (and K+) ionophores. The results suggest that in Halobacterium halobium, the binding site and thus the retinal Schiff base are exposed to the outside of the cell. This conclusion is consistent with a pumping mechanism based on a light-induced pK change.     

Effects of modifications of the retinal beta-ionone ring on archaebacterial sensory rhodopsin I.

Ring desmethyl and acyclic analogues of all-trans retinal were incorporated into the apoprotein of the phototaxis receptor sensory rhodopsin I (SR-I) in Halobacterium halobium membranes. All modified retinals generate SR-I analogue pigments which exhibit "opsin shifts," i.e., their absorption spectra are shifted to longer wavelengths compared with model protonated Schiff bases of the same analogues. Each SR-I pigment analogue exhibits cyclic photochemical reactions as monitored by flash spectroscopy, but the analogue photocycles differ from that of native SR-I by exhibiting pronounced biphasic recovery of flash-induced absorption changes and abnormal flash-induced absorption difference spectra. Despite perturbations in the photochemical properties, the SR-I pigment analogues are capable of both attractant (single photon) and repellent (two photon) phototaxis signaling in cells. Our interpretation is that the hydrophobic ring substituents interact with the binding pocket to maintain the correct configuration for native SR-I absorption and photochemistry, but these interactions are not essential for the physiological function of SR-I as a dual attractant/repellent phototaxis receptor. These results support the conclusion emerging from several studies that the photoactivation process that triggers the conformation changes of SR-I and the related proton pump bacteriorhodopsin is conserved despite the different biological functions of their photoactivation.     

Molecular dynamics study of the 13-cis form (bR548) of bacteriorhodopsin and its photocycle.

The structure and the photocycle of bacteriorhodopsin (bR) containing 13-cis,15-syn retinal, so-called bR548, has been studied by means of molecular dynamics simulations performed on the complete protein. The simulated structure of bR548 was obtained through isomerization of in situ retinal around both its C13-C14 and its C15-N bond starting from the simulated structure of bR568 described previously, containing all-trans,15-anti retinal. After a 50-ps equilibration, the resulting structure of bR548 was examined by replacing retinal by analogues with modified beta-ionone rings and comparing with respective observations. The photocycle of bR548 was simulated by inducing a rapid 13-cis,15-anti-->all-trans,15-syn isomerization through a 1-ps application of a potential that destabilizes the 13-cis isomer. The simulation resulted in structures consistent with the J, K, and L intermediates observed in the photocycle of bR548. The results offer an explanation of why an unprotonated retinal Schiff base intermediate, i.e., an M state, is not formed in the bR548 photocycle. The Schiff base nitrogen after photoisomerization of bR548 points to the intracellular rather than to the extracellular site. The simulations suggest also that leakage from the bR548 to the bR568 cycle arises due to an initial 13-cis,15-anti-->all-trans,15-anti photoisomerization.     

Factors affecting the C = N stretching in protonated retinal Schiff base: a model study for bacteriorhodopsin and visual pigments

Factors affecting the C = N stretching frequency of protonated retinal Schiff base (RSBH+) were studied with a series of synthetic chromophores and measured under different conditions. Interaction of RSBH+ with nonconjugated positive charges in the vicinity of the ring moiety or a planar polyene conformation (in contrast to the twisted retinal conformation in solution) shifted the absorption maxima but did not affect the C = N stretching frequency. The latter, however, was affected by environmental perturbations in the vicinity of the Schiff base linkage. Diminished ion pairing (i.e., of the positively charged nitrogen to its anion) achieved either by substituting a more bulky counteranion or by designing models with a homoconjugation effect lowered the C = N stretch energy. Decreasing solvation of the positively charged nitrogen leads to a similar trend. These effects in the vicinity of the Schiff base linkage also perturb the deuterium isotope effect observed upon deuteriation of the Schiff base. The results are interpreted by considering the mixing of the C = N stretching and C = N-H bending vibration. The C = N mode is shifted due to electrostatic interaction with nonconjugated positive charges in the vicinity of the Schiff base linkage, an interaction that does not influence the isotope effect. Weak hydrogen bonding between the Schiff base linkage in bacteriorhodopsin (bR) and its counteranion or, alternatively, poor solvation of the positively charged Schiff base nitrogen can account for the C = N stretching frequency of 1640 cm-1 and the deuterium isotope effect of 17 cm-1 observed in this pigment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analogue pigment studies of chromophore-protein interactions in metarhodopsins

Several analogue pigments have been prepared containing retinals altered at the cyclohexyl ring or proximal to the aldehyde group in order to examine the role of the chromophore in the formation of the metarhodopsin I and II states of visual pigments. Deletion of the 13-methyl group on the isoprenoid chain did not affect metarhodopsin formation. However, analogue pigments containing chromophores with modified rings did not show the typical absorption changes associated with the metarhodopsin transitions of native or regenerated rhodopsins. In particular, 4-hydroxyretinal pigments did not show clear transitions between the metarhodopsin I and metarhodopsin II states. Pigment formed with an acyclic retinal showed no evidence by absorption spectroscopy of metarhodopsin formation. A retinal altered by substitution of a five-membered ring containing a nitroxide required a more acidic pH than the native pigment for formation of the metarhodopsin II state. ESR data suggest that the ring remains buried within the protein through the metarhodopsin II state. However, the Schiff base linkage is susceptible to hydrolysis of hydroxylamine in the metarhodopsin II state. These data indicate that (1), in the transition from rhodopsin to metarhodopsin II, major protein conformational changes are occurring near the lysine-retinal linkage whereas the ring portion of the chromophore remains deeply buried within the protein and (2) pigment absorptions characteristic of the metarhodopsin I and II states may be due to specific protein-chromophore interactions near the region of the chromophore ring.     

Retinal isomerization in bacteriorhodopsin is controlled by specific chromophore-protein interactions. A study with noncovalent artificial pigments.

It has previously been shown that, in mutants lacking the Lys-216 residue, protonated Schiff bases of retinal occupy noncovalently the bacteriorhodopsin (bR) binding site. Moreover, the retinal-Lys-216 covalent bond is not a prerequisite for initiating the photochemical and proton pump activity of the pigment. In the present work, various Schiff bases of aromatic polyene chromophores were incubated with bacterioopsin to give noncovalent pigments that retain the Lys-216 residue in the binding site. It was observed that the pigment's absorption was considerably red-shifted relative to the corresponding protonated Schiff bases (PSB) in solution and was sensitive to Schiff base linkage substitution. Their PSB pK(a) is considerably elevated, similarly to those of related covalently bound pigments. However, the characteristic low-pH purple to blue transition is not observed, but rather a chromophore release from the binding site takes place that is characterized by a pK(a) of approximately 6 (sensitive to the specific complex). It is suggested that, in variance with native bR, in these complexes Asp-85 is protonated and Asp-212 serves as the sole negatively charged counterion. In contrast to the bound analogues, no photocycle could be detected. It is suggested that a specific retinal-protein geometrical arrangement in the binding site is a prerequisite for achieving the selective retinal photoisomerization.     

Femtosecond and picosecond fluorescence of native bacteriorhodopsin and a nonisomerizing analog

The spectrally and temporally resolved fluorescence properties of native bacteriorhodopsin (bR) and bR reconstituted with a nonisomerizing analog of the retinal Schiff base (bR5.12) are examined. The first attempt to experimentally monitor the excited state relaxation processes in both type of pigments using ultrafast fluorescence spectroscopy is reported. The fluorescence is emitted from retinal molecules in an all-trans configuration. Substantial energy relaxation involves very fast intramolecular and intermolecular vibrational modes and these are shown to occur on a time scale faster than isomerization. The possible contribution of dielectric interaction between the retinal Schiff base and the protein environment for the excited state energy relaxation is discussed.     

Non-Isomerizable Artificial Pigments: Implications for the Primary Light-Induced Events in Bacteriorhodopsin

The primary events in the photosynthetic retinal protein bacteriorhodopsin (bR) are reviewed in light of photophysical and photochemical experiments with artificial bR in which the native retinal polyene is replaced by a variety of chromophores. Focus is on retinals in which the critical C₁₃=C₁₄ bond is locked with respect to isomerization by a rigid ring structure. Other systems include retinal oxime and non-isomerizable dyes noncovalently residing in the binding site. The early photophysical events are analyzed in view of recent pump–probe experiments with sub-picosecond time resolution comparing the behavior of bR pigments with those of model protonated Schiff bases in solution. An additional approach is based on the light-induced cleavage of the protonated Schiff base bond that links retinal to the protein by reacting with hydroxylamine. Also described are EPR experiments monitoring reduction and oxidation reactions of a spin label covalently attached to various protein sites. It is concluded that in bR the initial relaxation out of the Franck–Condon (FC) state does not involve sub-stantial C₁₃=C₁₄ torsional motion and is considerably catalyzed by the protein matrix. Prior to the decay of the relaxed fluorescent state (FS or I state), the protein is activated via a mechanism that does not require double bond isomerization. Most plausibly, it is a result of charge delocalization in the excited state of the polyene (or other) chromophores. More generally, it is concluded that proteins and other macromolecules may undergo structural changes (that may affect their chemical reactivity) following optical excitation of an appropriately (covalently or non-covalently) bound chromophore. Possible relations between the light-induced changes due to charge delocalization, and those associated with C₁₃=C₁₄ isomerization (that are at the basis of the bR photocycle), are discussed. It is suggested that the two effects may couple at a certain stage of the photocycle, and it is the combination of the two that drives the cross-membrane proton pump mechanism.     

Mechanism of proton pumping in bacteriorhodopsin by solid-state NMR: the protonation state of tyrosine in the light-adapted and M states.

Solid-state 13C NMR spectra were employed to characterize the protonation state of tyrosine in the light-adapted (bR568) and M states of bacteriorhodopsin (bR). Difference spectra (isotopically labeled bR minus natural-abundance bR) were obtained for [4'-13C]Tyr-labeled bR, regenerated with [14-13C]retinal as an internal marker to identify the photocycle states. The [14-13C]retinal has distinct chemical shifts for bR555, for bR568, and for the M intermediate generated and thermally trapped at pH 10 in the presence of 0.3 M KCl or 0.5 M guanidine. Previous work has demonstrated that tyrosine and tyrosinate are easily distinguished on the basis of the chemical shift of the 4'-13C label and that both NMR signals are detectable in dark-adapted bR, although the tyrosinate signal is only present at pH values greater than 12. In the present work, we show that neither the light-adapted form of bR prepared at pH 7 or 10 nor the M state thermally trapped at -80 degrees C in 0.3 M KCl pH 10, or in 0.5 M guanidine pH 10, shows any detectable tyrosinate. In addition, after the M samples were briefly warmed (approximately 30 s), no tyrosinate was observed. However, small (1-2 ppm) changes in the structure or dispersion in the Tyr peak were observed in the M state phototrapped by either method. These changes were reversible when the sample was warmed, although on a time scale slower than the relaxation of the retinal back to the bR568 conformer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solid-state 13C and 15N NMR study of the low pH forms of bacteriorhodopsin

The visible absorption of bacteriorhodopsin (bR) is highly sensitive to pH, the maximum shifting from 568 nm (pH 7) to approximately 600 nm (pH 2) and back to 565 nm (pH 0) as the pH is decreased further with HCl. Blue membrane (lambda max greater than 600 nm) is also formed by deionization of neutral purple membrane suspensions. Low-temperature, magic angle spinning 13C and 15N NMR was used to investigate the transitions to the blue and acid purple states. The 15N NMR studies involved [epsilon-15N]lysine bR, allowing a detailed investigation of effects at the Schiff base nitrogen. The 15N resonance shifts approximately 16 ppm upfield in the neutral purple to blue transition and returns to its original value in the blue to acid purple transition. Thus, the 15N shift correlates directly with the color changes, suggesting an important contribution of the Schiff base counterion to the "opsin shift". The results indicate weaker hydrogen bonding in the blue form than in the two purple forms and permit a determination of the contribution of the weak hydrogen bonding to the opsin shift at a neutral pH of approximately 2000 cm-1. An explanation of the mechanism of the purple to blue to purple transition is given in terms of the complex counterion model. The 13C NMR experiments were performed on samples specifically 13C labeled at the C-5, C-12, C-13, C-14, or C-15 positions in the retinylidene chromophore. The effects of the purple to blue to purple transitions on the isotropic chemical shifts for the various 13C resonances are relatively small. It appears that bR600 consists of at least four different species. The data confirm the presence of 13-cis- and all-trans-retinal in the blue form, as in neutral purple dark-adapted bR. All spectra of the blue and acid purple bR show substantial inhomogeneous broadening which indicates additional irregular distortions of the protein lattice. The amount of distortion correlates with the variation of the pH, and not with the color change.     

Effective light-induced hydroxylamine reactions occur with C13 = C14 nonisomerizable bacteriorhodopsin pigments.

The light-driven proton pump bacteriorhodopsin (bR) undergoes a bleaching reaction with hydroxylamine in the dark, which is markedly catalyzed by light. The reaction involves cleavage of the (protonated) Schiff base bond, which links the retinyl chromophore to the protein. The catalytic light effect is currently attributed to the conformational changes associated with the photocycle of all-trans bR, which is responsible for its proton pump mechanism and is initiated by the all-trans --> 13-cis isomerization. This hypothesis is now being tested in a series of experiments, at various temperatures, using three artificial bR molecules in which the essential C13==C14 bond is locked by a rigid ring structure into an all-trans or 13-cis configuration. In all three cases we observe an enhancement of the reaction by light despite the fact that, because of locking of the C13==C14 bond, these molecules do not exhibit a photocycle, or any proton-pump activity. An analysis of the rate parameters excludes the possibility that the light-catalyzed reaction takes place during the approximately 20-ps excited state lifetimes of the locked pigments. It is concluded that the reaction is associated with a relatively long-lived (micros-ms) light-induced conformational change that is not reflected by changes in the optical spectrum of the retinyl chromophore. It is plausible that analogous changes (coupled to those of the photocycle) are also operative in the cases of native bR and visual pigments. These conclusions are discussed in view of the light-induced conformational changes recently detected in native and artificial bR with an atomic force sensor.     

Functional interactions in bacteriorhodopsin: a theoretical analysis of retinal hydrogen bonding with water.

The light-driven proton pump, bacteriorhodopsin (bR) contains a retinal molecule with a Schiff base moiety that can participate in hydrogen-bonding interactions in an internal, water-containing channel. Here we combine quantum chemistry and molecular mechanics techniques to determine the geometries and energetics of retinal Schiff base-water interactions. Ab initio molecular orbital calculations are used to determine potential surfaces for water-Schiff base hydrogen-bonding and to characterize the energetics of rotation of the C-C single bond distal and adjacent to the Schiff base NH group. The ab initio results are combined with semiempirical quantum chemistry calculations to produce a data set used for the parameterization of a molecular mechanics energy function for retinal. Using the molecular mechanics force field the hydrated retinal and associated bR protein environment are energy-minimized and the resulting geometries examined. Two distinct sites are found in which water molecules can have hydrogen-bonding interactions with the Schiff base: one near the NH group of the Schiff base in a polar region directed towards the extracellular side, and the other near a retinal CH group in a relatively nonpolar region, directed towards the cytoplasmic side.     

Catalysis of the retinal subpicosecond photoisomerization process in acid purple bacteriorhodopsin and some bacteriorhodopsin mutants by chloride ions.

The dynamics and the spectra of the excited state of the retinal in bacteriorhodopsin (bR) and its K-intermediate at pH 0 was compared with that of bR and halorhodopsin at pH 6.5. The quantum yield of photoisomerization in acid purple bR was estimated to be at least 0.5. The change of pH from 6.5 to 2 causes a shift of the absorption maximum from 568 to 600 nm (acid blue bR) and decreases the rate of photoisomerization. A further decrease in pH from 2 to 0 shifts the absorption maximum back to 575 nm when HCl is used (acid purple bR). We found that the rate of photoisomerization increases when the pH decreases from 2 to 0. The effect of chloride anions on the dynamics of the retinal photoisomerization of acid bR (pH 2 and 0) and some mutants (D85N, D212N, and R82Q) was also studied. The addition of 1 M HCl (to make acid purple bR, pH 0) or 1 M NaCl to acid blue bR (pH 2) was found to catalyze the rate of the retinal photoisomerization process. Similarly, the addition of 1 M NaCl to the solution of some bR mutants that have a reduced rate of retinal photoisomerization (D85N, D212N, and R82Q) was found to catalyze the rate of their retinal photoisomerization process up to the value observed in wild-type bR. These results are explained by proposing that the bound Cl- compensates for the loss of the negative charges of the COO- groups of Asp85 and/or Asp212 either by neutralization at low pH or by residue replacement in D85N and D212N mutants.