Photochemistry of two rhodopsinlike pigments in bacteriorhodopsin-free mutant of Halobacterium halobium.

Two photocycles due to two different pigments were found in membrane vesicles of a bacteriorhodopsin-free mutant of Halobacterium halobium. A pigment absorbing approximately 590 nm halorhodopsin (HR) underwent a faster photocycle with a phototransient at approximately 490 nm (half-time of decay, tau 1/2 = 10 ms). Another third rhodopsinlike pigment (TR) absorbing approximately 580 nm underwent a slower photocycle accompanying a phototransient absorbing below 410 nm (tau 1/2 = 0.8s). The photocycles were measured under various conditions of temperature, NaCl concentration, pH, and in the presence of cholate. All results obtained support the notion that the two photocycles are independent of each other, and the fast or the slow cycle can be abolished after these treatments. At alkaline pH, the wavelength of maximum absorbance of both pigments shifted to blue, but the magnitude of the shift of the pigment undergoing the slow photocycle was much greater than the other. The ratio of the content of the two pigments varies among bacteriorhodopsin-free mutants.     

The photocycle of the chloride pump halorhodopsin. I: Azide-catalyzed deprotonation of the chromophore is a side reaction of photocycle intermediates inactivating the pump

Halorhodopsin, the light-driven chloride pump of halobacteria, undergoes a photochemical cycle in the 10 ms range. Two intermediates, HR640 and HR520, accumulate in the photosteady state after short times (within 100 ms) of illumination. Upon prolonged illumination a third species, HRL410 accumulates, which is formed from HR520/HR640 by deprotonation of the chromophore in a side reaction of the photocycle. In the dark, HRL410 requires several minutes to reconvert thermally to HR478. Thus, molecules in the HRL410 state must be inactive pumps since their maximal turnover number could only be a few per hour. Inorganic bases, such as azide, catalyze the deprotonation of HR520/HR640 as well as the reprotonation of HRL410. Both reactions are accelerated several hundred times by azide but the photosteady-state concentration of HRL410 remains unchanged.     

Transient spectroscopy of bacterial rhodopsins with an optical multichannel analyzer. 1. Comparison of the photocycles of bacteriorhodopsin and halorhodopsin

We used a gated optical multichannel analyzer to measure transient flash-induced absorption changes in bacteriorhodopsin (BR) and halorhodopsin (HR) and developed criteria for calculating the absorption spectra of the photocycle intermediates and the kinetics of their rise and decay. The results for BR agree with data reported by a large number of other authors. The results for HR in the presence of chloride are consistent with earlier data and reveal an additional intermediate, not previously seen, in the submicrosecond time scale. Although an M412-like intermediate is not in the HR photocycle, a one-by-one comparison of the rest of the intermediates observed for BR and HR indicates a striking similarity between the photocycles of the two bacterial rhodopsins. This was previously not apparent, perhaps because the experimental approaches to the spectroscopy of the two pigments were different and the data were thus more fragmented.     

Characterization of the azide-dependent bacteriorhodopsin-like photocycle of salinarum halorhodopsin

The photocycle of salinarum halorhodopsin was investigated in the presence of azide. The azide binds to the halorhodopsin with 150 mM binding constant in the absence of chloride and with 250 mM binding constant in the presence of 1 M chloride. We demonstrate that the azide-binding site is different from that of chloride, and the influence of chloride on the binding constant is indirect. The analysis of the absorption kinetic signals indicates the existence of two parallel photocycles. One belongs to the 13-cis retinal containing protein and contains a single red shifted intermediate. The other photocycle, of the all-trans retinal containing halorhodopsin, resembles the cycle of bacteriorhodopsin and contains a long-living M intermediate. With time-resolved spectroscopy, the spectra of intermediates were determined. Intermediates L, N, and O were not detected. The multiexponential rise and decay of the M intermediate could be explained by the introduction of the "spectrally silent" intermediates M1, M2, and HR', HR, respectively. The electric signal measurements revealed the existence of a component equivalent with a proton motion toward the extracellular side of the membrane, which appears during the M1 to M2 transition. The differences between the azide-dependent photocycle of salinarum halorhodopsin and pharaonis halorhodopsin are discussed.     

Photocycle of halorhodopsin from Halobacterium salinarium.

The light-driven chloride pump, halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-adapted conditions and can exist in chloride-free and chloride-binding forms. To describe the photochemical cycle of the all-trans, chloride-binding state that is associated with the transport, and thereby initiate study of the chloride translocation mechanism, one must first dissect the contributions of these species to the measured spectral changes. We resolved the multiple photochemical reactions by determining flash-induced difference spectra and photocycle kinetics in halorhodopsin-containing membranes prepared from Halobacterium salinarium, with light- and dark-adapted samples at various chloride concentrations. The high expression of cloned halorhodopsin made it possible to do these measurements with unfractionated cell envelope membranes in which the chromophore is photostable not only in the presence of NaCl but also in the Na2SO4 solution used for reference. Careful examination of the flash-induced changes at selected wavelengths allowed separating the spectral changes into components and assigning them to the individual photocycles. According to the results, a substantial revision of the photocycle model for H. salinarium halorhodopsin, and its dependence on chloride, is required. The cycle of the all-trans chloride-binding form is described by the scheme, HR-hv-->K<==>L1<==>L2<==>N-->HR, where HR, K, L, and N designate halorhodopsin and its photointermediates. Unlike the earlier models, this is very similar to the photoreaction of bacteriorhodopsin when deprotonation of the Schiff base is prevented (e.g., at low pH or in the D85N mutant). Also unlike in the earlier models, no step in this photocycle was noticeably affected when the chloride concentration was varied between 20 mM and 2 M in an attempt to identify a chloride-binding reaction.     

Properties and photochemistry of a halorhodopsin from the haloalkalophile, Natronobacterium pharaonis

Pharaonis halorhodopsin is a light-driven transport system for chloride, similarly to the previously described halorhodopsin, but we find that it transports nitrate as effectively as chloride. We studied the photoreactions of the purified, detergent-solubilized pharaonis pigment with a gated multichannel analyzer. At a physiological salt concentration (4 M NaCl), the absorption spectra and rate constants of rise and decay for intermediates of the photocycle were similar to those for halorhodopsin. In buffer containing nitrate, halorhodopsin exhibits a second, truncated photocycle; this difference in the photoreaction of the pigment occurs when an anion is bound in such a way as to preclude transport. As expected from the lack of anion specificity in the transport, the photocycle of pharaonis halorhodopsin was nearly unaffected by replacement of chloride with nitrate. All presumed buried positively charged residues, which might play a role in anion binding, are conserved in the two pigments. At the extracellular end of the presumed helix C, however, an arginine residue is found in halorhodopsin, but not in pharaonis halorhodopsin, and an arginine-rich segment between the presumed helices A and B in halorhodopsin is replaced by a less positively charged sequence in pharaonis halorhodopsin (Lanyi, J. K., Duschl, A., Hatfield, G. W., May, K., and Oesterhelt, D. (1990) J. Biol. Chem. 265, 1253-1260). One or both of these alterations may explain the difference in the anion selectivity of the two proteins.     

Anion uptake in halorhodopsin from Natromonas pharaonis studied by FTIR spectroscopy: consequences for the anion transport mechanism

The uptake of chloride, bromide, iodide, nitrate, and azide by anion-depleted blue halorhodopsin from Natronobacterium pharaonis has been followed by FTIR difference spectroscopy using an ATR sampling device. The spectra are compared with the spectrum of the O intermediate obtained by time-resolved FTIR studies of the photocycle. It is demonstrated that anion-free blue halorhodopsin can be identified with the O intermediate and, thus, that the decay of O is due to the passive uptake of the anion. The great similarity of the anion-binding spectra and their identity in the case of the monoatomic anions indicate a rather unspecific binding site for the different anions dominated by electrostatic interactions. Comparing spectra obtained with 15N nitrate and unlabeled nitrate, the NO-stretching bands could be identified. The small splitting and the small IR intensity of those bands indicate a rather nonpolar binding site with a rather isotropic influence on the nitrate, in contrast to aqueous nitrate. In further experiments on the photocycle of blue halorhodopsin, the all-trans --> 13-cis isomerization can be clearly identified. Up to 100 micros, the isomerization-induced structural changes deduced from amide I changes are similar to those occurring during the anion-transporting photocycle. Compared to these, the molecular changes involved in the release and their reversion during the uptake of anions are considerably larger. They can be reached via two pathways: (1) by reducing the anion concentration and (2) transiently during the anion-transporting photocycle with the formation of the precursor of O with O conformation. Consequences of the anion transport mechanism are discussed.     

Photoreactions of bacteriorhodopsin at acid pH.

It has been known that bacteriorhodopsin, the retinal protein in purple membrane which functions as a light-driven proton pump, undergoes reversible spectroscopic changes at acid pH. The absorption spectra of various bacteriorhodopsin species were estimated from measured spectra of the mixtures that form at low pH, in the presence of sulfate and chloride. The dependency of these on pH and the concentration of Cl- fit a model in which progressive protonation of purple membrane produces "blue membrane", which will bind, with increasing affinity as the pH is lowered, chloride ions to produce "acid purple membrane." Transient spectroscopy with a multichannel analyzer identified the intermediates of the photocycles of these altered pigments, and described their kinetics. Blue membrane produced red-shifted KL-like and L-like products, but no other photointermediates, consistent with earlier suggestions. Unlike others, however, we found that acid purple membrane exhibited a very different photocycle: its first detected intermediate was not like KL in that it was much more red-shifted, and the only other intermediate detectable resembled the O species of the bacteriorhodopsin photocycle. An M-like intermediate, with a deprotonated Schiff base, was not found in either of these photocycles. There are remarkable similarities between the photoreactions of the acid forms of bacteriorhodopsin and the chloride transport system halorhodopsin, where the Schiff base deprotonation seems to be prevented by lack of suitable aspartate residues, rather than by low pH.     

Effect of anions on the photocycle of halorhodopsin. Substitution of chloride with formate anion

Halorhodopsin from Natronomonas pharaonis is a light-driven chloride pump which transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. It was shown that the chloride anion bound in the vicinity of retinal PSB can be replaced by several inorganic anions, including azide which converts the chloride pump into a proton pump and induces formation of an M-like intermediate detected in the bR photocycle but not in native halorhodopsin. Here we have studied the possibility of replacing the chloride anion with organic anions and have followed the photocycle under several conditions. It is revealed that the chloride can be replaced with a formate anion but not with larger organic anions such as acetate. Flash photolysis experiments detected in the formate pigment an M-like intermediate characterized by a lifetime much longer than that of the O intermediate. The lifetime of the M-like intermediate depends on the pH, and its decay is significantly accelerated at low pH. The decay rate exhibited a titration-like curve, suggesting that the protonation of a protein residue controls the rate of M decay. Similar behavior was detected in N. pharaonis pigments in which the chloride anion was replaced with NO(2)(-) and OCN(-) anions. It is suggested that the formation of the M-like intermediate indicates branching pathways from the L intermediate or basic heterogeneity in the original pigment.     

Flash spectroscopic studies of the kinetics of the halorhodopsin photocycle

The photoreactions of halorhodopsin are complicated by the fact that the parent pigment and its photoproducts interact with chloride. Thus, in any photoreaction scheme at least four species have to be accounted for: HR565 and HR578 Cl-, as well as HR640 and HR520 Cl-. A photocycle scheme proposed earlier places the two main photointermediates of halorhodopsin, HR520 Cl- and HR640, into a single photocycle, with a chloride-dependent equilibrium between them [Oesterhelt, D., Hegemann, P., & Tittor, J. (1985) EMBO J. 4, 2351-2356]. This scheme, with the additional feature of direct photoproduction of HR640 from HR565, was tested in this work by using numerical solutions of the appropriate differential equations to simulate flash-induced absorption changes at 500 nm (production of HR520 Cl-) and at 660 nm (production of HR640). The time scale of the simulation was ms following the flash. Comparison of the simulated curves with experimental traces yielded a unique set of three rate constants. The proposed photocycle scheme and these rate constants predict well the shapes and amplitudes of flash traces at various chloride concentrations. It appears from the photocycle scheme, and the numerical values of rate constants, that chloride is bound with high affinity to the parent halorhodopsin molecule, but with much lower affinity to its main photointermediate. This may be the consequence of the fact that in the parent halorhodopsin in the retinal configuration is all-trans, but in the two photointermediates it is 13-cis.     

Mechanism of a molecular valve in the halorhodopsin chloride pump

Halorhodopsin is a light-driven chloride anion pump in which the trans-->cis photoisomerization of a retinal chromophore triggers a photocycle resulting in the translocation of chloride across the plasma membrane. The mechanism of chloride transfer past the cis retinal is determined here by computing multiple pathways for this process. The calculations reveal two conditions of the valve mechanism. First, a lumen absent in the ground state structure is transiently opened by chloride passage. Second, this activated opening, which is achieved by flexible deformation of the surrounding protein, is shown to significantly raise the chloride translocation barrier between photocycles, thus preventing chloride backflow. Unlike macroscopic valve designs, the protein allows differential ion flows in the pumping and resting states that are tuned to match the physiological timescales of the cell, thus creating a "kinetic" valve.     

Effects of anion binding on the deprotonation reactions of halorhodopsin

The retinal Schiff base of halorhodopsin deprotonates with a pKa of 7.4 in 0.5 M Na2SO4 in the dark. In the presence of various anions, such as chloride or nitrate, etc., the pKa is raised by up to 1.5 units. Analysis of the dependency of the pKa on anion concentration favors the model in which the anions do not bind to the positively charged Schiff base nitrogen, but to a site near it, and exert their effect on the pKa by direct (perhaps electrostatic) interaction. Adding nitrate, or one of several other anions, causes also a small blueshift in the visible absorption band of the chromophore. These effects on the pKa and the absorption band define an anion binding site in halorhodopsin, termed Site I. Chloride and bromide apparently bind in addition to another site, which is associated with a small red-shift of the absorption band and changes in the photocycle. This other anion binding site is termed Site II. Illumination of halorhodopsin samples results in the deprotonation of the Schiff base with a much lowered pKa, but at very low rates probably determined by the generation of a deprotonating photointermediate. Binding of Site I anions increases the pKa of deprotonation in the light also. The similarity of the responses of the apparent pKa in the dark and in the light to anion concentration suggests that anion binding to Site I influences deprotonation of the Schiff base similarly in the photointermediate and in the parent halorhodopsin molecule.     

The nitrate transporting photochemical reaction cycle of the pharaonis halorhodopsin

Time-resolved spectroscopy, absorption kinetic and electric signal measurement techniques were used to study the nitrate transporting photocycle of the pharaonis halorhodopsin. The spectral titration reveals two nitrate-binding constants, assigned to two independent binding sites. The high-affinity binding site (K(a) = 11 mM) contributes to the appearance of the nitrate transporting photocycle, whereas the low-affinity constant (having a K(a) of approximately 7 M) slows the last decay process in the photocycle. Although the spectra of the intermediates are not the same as those found in the chloride transporting photocycle, the sequence of the intermediates and the energy diagrams are similar. The differences in spectra and energy levels can be attributed to the difference in the size of the transported chloride or nitrate. Electric signal measurements show that a charge is transferred across the membrane during the photocycle, as expected. A new observation is an apparent release and rebinding of a small fraction of the retinal, inside the retinal pocket, during the photocycle. The release occurs during the N-to-O transition, whereas the rebinding happens in several seconds, well after the other steps of the photocycle are over.     

M-decay in the bacteriorhodopsin photocycle: effect of cooperativity and pH

The dependence of the bacteriorhodopsin (bR) photocycle on the intensity of the exciting flash was investigated in purple membranes. The dependence was most pronounced at slightly alkaline pH values. A comparison study of the kinetics of the photocycle and proton uptake at different intensities of the flash suggested that there exist two parallel photocycles in purple membranes at a high intensity of the flash. The photocycle of excited bR in a trimer with the two other bR molecules nonexcited is characterized by an almost irreversible M --> N transition. Excitation of two or three bR in a trimer induces the N --> M back reaction and accelerates the N --> bR transition. Based on the qualitative similarity of the pH dependencies of the photocycles of solubilized bR and excited dimers and trimers we proposed that the interaction of nonexcited bR in trimers alters the photocycle of the excited monomer as compared to solubilized bR and the changes in the photocycles in excited dimers and trimers are the result of decoupling of this interaction.     

Low-temperature photoreactions of halorhodopsin. 2. Description of the photocycle and its intermediates

Photostationary states of halorhodopsin (HR, a retinal protein in the halobacterial membrane) and their further thermal conversions were investigated at 140-230 K by absorption spectroscopy in the visible. The difference spectra confirm several steps of the all-trans-HR photocycle, in the presence of chloride, proposed earlier on the basis of room temperature flash spectroscopy. Thus, at 140 K, the spectra reveal the HR600----HR520 reaction, and at 170-230 K the HR640----HR578 and the HR520----HR578 reactions can be seen. No evidence for the expected HR520 in equilibrium HR640 process was found, however. From the difference spectra at various temperatures, exact absorption spectra of HR600 and HR520 were calculated, and an estimate of the HR640 spectrum in a mixture also containing HR520 was obtained. The low-temperature absorption maxima of HR578 and its photointermediates relate to the room temperature maxima differently from what is expected from the spectra of the corresponding intermediates in the bacteriorhodopsin photocycle.     

Influence of the Charge at D85 on the Initial Steps in the Photocycle of Bacteriorhodopsin

Studies have shown that trans-cis isomerization of retinal is the primary photoreaction in the photocycle of the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum, as well as in the photocycle of the chloride pump halorhodopsin (HR). The transmembrane proteins HR and BR show extensive structural similarities, but differ in the electrostatic surroundings of the retinal chromophore near the protonated Schiff base. Point mutation of BR of the negatively charged aspartate D85 to a threonine T (D85T) in combination with variation of the pH value and anion concentration is used to study the ultrafast photoisomerization of BR and HR for well-defined electrostatic surroundings of the retinal chromophore. Variations of the pH value and salt concentration allow a switch in the isomerization dynamics of the BR mutant D85T between BR-like and HR-like behaviors. At low salt concentrations or a high pH value (pH 8), the mutant D85T shows a biexponential initial reaction similar to that of HR. The combination of high salt concentration and a low pH value (pH 6) leads to a subpopulation of 25% of the mutant D85T whose stationary and dynamic absorption properties are similar to those of native BR. In this sample, the combination of low pH and high salt concentration reestablishes the electrostatic surroundings originally present in native BR, but only a minor fraction of the D85T molecules have the charge located exactly at the position required for the BR-like fast isomerization reaction. The results suggest that the electrostatics in the native BR protein is optimized by evolution. The accurate location of the fixed charge at the aspartate D85 near the Schiff base in BR is essential for the high efficiency of the primary reaction.     

Transport of anions in isolated barley vacuoles : I. Permeability to anions and evidence for a cl-uptake system

The anion contents of young barley leaves and of mesophyll protoplasts from the leaves was compared. Anion loss from the protoplasts during isolation was small. Although only about 60% of the leaf cells were mesophyll cells, phosphate and sulfate contents of the mesophyll cells accounted for almost 90% of the leaf contents. Chloride accumulated in the leaf epidermis. The rapid isolation of vacuoles from mesophyll protoplasts permitted the determination of vacuolar ion concentrations. Sodium and nitrate levels were very low in the cytoplasm, and much higher in the vacuole. When barley plants were grown in the presence of low NaCl levels, chloride concentrations were comparable in cytoplasm and vacuole, and similar observations were made with sulfate. Cytoplasmic phosphate concentrations were close to 30 millimolar and potassium concentrations 100 millimolar. During a 30 minute incubation period at room temperature, anion contents of isolated vacuoles decreased considerably. Efflux of NO₃⁻ was faster than that of Cl⁻. Phosphate and sulfate crossed the tonoplast only slowly. 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid partially inhibited the efflux of nitrate and, to a lesser extent, that of chloride. Decreased efflux was also observed in the presence of MgATP. In remarkable contrast, p-chloromercuribenzene sulfonate and HgCl₂ stimulated the efflux of nitrate and chloride, but not of phosphate. Labeled chloride was taken up by isolated vacuoles. The apparent K_m for chloride uptake at low chloride concentrations was 2.3 millimolar. At elevated chloride concentrations, chloride did not display saturation characteristics but, rather, characteristics of a diffusional process. Uptake was stimulated by ATP.     

Photocycles of bacteriorhodopsin in light- and dark-adapted purple membrane studied by time-resolved absorption spectroscopy.

Nanosecond time-resolved absorption spectra have been measured throughout the photocycle of bacteriorhodopsin in both light-adapted and dark-adapted purple membrane (PM). The data from dark-adapted samples are interpretable as the superposition of two photocycles arising independently from the all-trans and 13-cis retinal isomers that coexist in the dark-adapted state. The presence of a photocycle in dark-adapted PM which is indistinguishable from that observed for light-adapted PM under the same experimental conditions is demonstrated by the observation of the same five relaxation rates associated with essentially identical changes in the photoproduct spectra. This cycle is attributed to the all-trans component. The cycle of the 13-cis component is revealed by scaling the data measured for the light-adapted sample and subtracting it from the data on the dark-adapted mixture. At times less than 1 ms, the resulting difference spectra are nearly time-independent. The peak of the difference spectrum is near 600 nm, although there appears to be a slight (approximately 2 nm) blue-shift in the first few microseconds. Subsequently the amplitude of this spectrum decays and the peak of the difference spectrum shifts in two relaxations. Most of the amplitude of the photoproduct difference spectrum (approximately 80%) decays in a single relaxation having a time constant of approximately 35 ms. The difference spectrum remaining after this relaxation peaks at approximately 590 nm and is indistinguishable from the classical light-dark difference spectrum, which we find, in experiments performed on a much longer time scale, to peak at 588 nm. The decay of this remaining photo-product is not resolvable in the nanosecond kinetic experiments, but dark adaptation of a completely light-adapted sample is found to occur exponentially with a relaxation time of approximately 2,000 s under the conditions of our experiments.     

Halide binding by the purified halorhodopsin chromoprotein. I. Effects on the chromophore

The halorhodopsin chromoprotein, a retinal-protein complex with an apparent molecular mass of 20 kilo-daltons, exhibits all of the halide-dependent effects found for the chromophore of functional halorhodopsin in cell envelope vesicles. With increasing halide concentration (a) an alkali-dependent 580/410 nm chromophore equilibrium (attributed to reversible deprotonation of the retinal Schiff's base) is shifted toward the 580-nm chromophore and (b) the flash-induced photocycle proceeds increasingly via P520, rather than via P660. The halide-binding site(s) responsible for these effects must reside, therefore, in the chromoprotein. Chloride and bromide are about equivalent, but iodide is much less effective in these effects and in being transported. Several other anions, i.e. thiocyanate, nitrate, phosphate, and acetate, affect the absorption maximum of the chromophore but do not allow the production of P520 upon flash illumination and are not transported. However, these ions appear to compete with chloride in the flash experiments. These observations suggest that binding of anions to a relatively nonspecific site affects the protonation state of the Schiff's base in the chromophore. Either this site directly or a more specific site, connected to the first one by a sequential pathway, is involved with the photocycle intermediates and with chloride transport by halorhodopsin.