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.     

Halide binding by the purified halorhodopsin chromoprotein. II. New chloride-binding sites revealed by 35Cl NMR

Halorhodopsin is a light-driven chloride pump in the cell membrane of Halobacterium halobium. Recently, a polypeptide of apparent Mr = 20,000 has been purified that contains the halorhodopsin chromophore. Here we use 35Cl NMR to show that the purified chromoprotein possesses two previously unknown classes of chloride-binding sites. One class exhibits a low affinity (KD much greater than 1 M) for chloride and bromide. The second class exhibits a higher affinity (KD = 110 +/- 50 mM) for chloride and also binds other anions according to the affinity series I-, SCN- greater than Br-, NO-3 greater than Cl- greater than F-, citrate. Both classes of NMR site remain intact at pH 11, indicating that the essential positive charges are provided by arginine. Also, both classes are unaffected by bleaching, suggesting that the sites are not in the immediate vicinity of the halorhodopsin chromophore. Although the chromoprotein also appears to contain the chloride-transport site (Steiner, M., Oesterhelt, D., Ariki, M., and Lanyi, J. K. (1984) J. Biol. Chem. 259, 2179-2184), this site was not detected by 35Cl NMR, suggesting that the transport site is in the interior of the protein where it is sampled slowly by chloride in the medium. It is proposed that the purified chromoprotein possesses a channel leading from the medium to the transport site and that the channel contains the high affinity NMR site which facilitates the migration of chloride between the medium and the transport site. We have also used 35Cl NMR to study chloride binding to purified monomeric bacteriorhodopsin; however, this protein contains no detectable chloride-binding sites.     

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.     

Properties and the primary structure of a new halorhodopsin from halobacterial strain mex.

A new halorhodopsin-like pigment from the new halobacterial strain mex (Otomo, J., Tomoika, H. and Sasabe, H. (1992) J. Gen. Microbiol. 138, 1027-1037) was partially purified, and its amino acid sequence from helices A to G was determined using PCR technique. Two arginine residues in the A-B interhelix loop segment, a series of six amino acid residues (EMPAGH) in the B-C interhelix segment and most of the residues near the Schiff base of the retinal were found to be conserved in three halorhodopsins (halobium, pharaonis and mex). This result strongly suggests that these residues are essential for anion pumping function in halorhodopsin. The light-induced ion-pump measurements have shown that the selectivity of anion transport between chloride and nitrate in mex halorhodopsin is lower than that of halobium halorhodopsin, but higher than that of pharaonis halorhodopsin. The number of amino acid residues in the B-C interhelix loop segments is different in each halorhodopsin, and it correlates with their anion (chloride and nitrate) selectivity. These results suggest that the length of the B-C segment affects the selectivity of anion transport in halorhodopsin.     

Effects of arginine modification on the photocycle of halorhodopsin

Exhaustive reaction with phenylglyoxal removed 9 of the 12 arginine and 1 of the 2 lysine residues in detergent-solubilized halorhodopsin, without affecting the chromophore. The consequences of this extensive removal of positive charges on various chloride-binding equilibria and the photochemistry were evaluated. No significant effects were seen on the affinity of Site I to chloride and on the increase in the pKa of Schiff-base deprotonation, which is caused by the chloride binding at this site. No significant effects were seen on the affinity of Site II to chloride, either. However, the photocycle of the pigment was affected. Kinetic modeling of the observed changes in flash-induced absorption changes suggests that the modification increases the affinity of the main halorhodopsin photointermediate to chloride by about fourfold. If chloride translocation involves release of chloride from this intermediate during the transport cycle, the result might explain the observed partial inhibitory effects on chloride transport. Plausible models of chloride translocation include reversible binding of the anion by positively charged groups, strategically arranged in the protein. The results indicate that two of the three spectroscopically observable chloride-dependent equilibria do not depend on a large number of positively charged residues in the protein. To the extent that the unaffected equilibria represent association and dissociation which occur during chloride translocation, at least part of the chloride translocation might be accomplished with the participation of only a few positively charged residues.     

Primary and secondary chloride transport in Halobacterium halobium.

Chloride uptake in intact cells of Halobacterium halobium was characterized by rates of influx and efflux of 36Cl- under conditions of light, respiration, or both. Halobacterial mutant strains with and without retinal transport proteins allowed study of the effects of halorhodopsin and bacteriorhodopsin under illumination. Two structurally independent chloride transport systems could be distinguished: halorhodopsin, the already known light-driven chloride pump, and a newly described secondary uptake system, which was energized by respiration or by light via bacteriorhodopsin.     

Light-dependent trans to cis isomerization of the retinal in halorhodopsin

Flash-induced absorption changes in the near UV were determined for bacteriorhodopsin and halorhodopsin on a millisecond time scale. The difference spectrum obtained for bacteriorhodopsin was comparable to model difference spectra of tyrosine (aromatic OH deprotonated vs protonated), as found by others. The flash-induced difference spectrum for halorhodopsin, in contrast, resembled a model spectrum obtained for trans to 13-cis isomerization of retinal in bacteriorhodopsin. A model for chloride translocation by halorhodopsin is presented, in which the retinal isomerization moves positive charges, which in turn modulate the affinity of a site to chloride.     

Two pumps, one principle: light-driven ion transport in halobacteria

Comparison of the primary structure of the chloride pump halorhodopsin with that of the proton pump bacteriorhodopsin provides insight into light-driven ion transport by retinal proteins. Several conserved amino acid residues in the membrane-spanning region of both proteins and their interaction with different isomerization states of retinal are suggested to be the key element for ion transport in both proteins.     

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.     

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.     

Functional reconstitution of halorhodopsin. Properties of halorhodopsin-containing proteoliposomes

A one-step purification method for halorhodopsin was developed. Functional proteoliposomes were prepared from this preparation using cholate, which is removed by dialysis in the presence of asolectin or the polar halobacterial lipids. Light-induced outward directed transport of chloride by halorhodopsin was followed by measuring passive proton efflux in the presence of uncoupler; initial rates and extents amounted to significant fractions of values obtained for halorhodopsin-containing cell envelope vesicles. The transport activity was much higher when cholate rather than octyl glucoside was used in the reconstitution. Since CD spectra in cholate but not in octyl glucoside showed band-splitting in the visible region, suggestive of exciton interaction between halorhodopsin monomers, the reconstitution may depend on an aggregate state of the halorhodopsin. The rate constants for three thermal steps in the halorhodopsin photocycle were greatly reduced in the detergent-solubilized samples, but they increased in the proteoliposomes to values similar to those for halorhodopsin in cell envelope vesicles. Thus, the reconstitution yields halorhodopsin with both photochemical and transport activities restored. Freeze-fracture electron micrographs of the proteoliposomes showed unilammellar liposomes with numerous particles of 100-150 A diameter at the fracture faces. These should correspond to halorhodopsin aggregates, formed in the bilayer in an apparently concentration-dependent manner.     

Specific arginine and threonine residues control anion binding and transport in the light-driven chloride pump halorhodopsin.

The light-driven chloride pump halorhodopsin (HR), a halobacterial retinal protein, was studied by comparing wild type with specific mutants. Changes of conserved arginine and threonine residues in the transmembrane regions could be classified in two categories: in the extracellular half of the molecule, mutations influence anion uptake and binding. R108 mutations abolish all anion effects previously attributed to two distinct binding sites and change the characteristic photochemistry. Neutral residues at position 108 completely inactivate the pump. T111 increases the affinity of this anion binding site without being essentially important. In the photochemical cycles of the mutants T111V and Q105E, a red-shifted absorbing intermediate is enriched indicating retarded anion uptake. On the cytoplasmic side, mutations do not change anion binding properties of the unphotolyzed protein, but slow down anion release thereby reducing the chloride transport activity and the photocycling rate. The lowest activity is found for T203V, while R200 mutations have weaker effects. Thus, in the symmetrically arranged pairs R108/T111 and T203/R200, threonine and arginine play different roles, reflecting high affinity anion uptake by the former and effective anion release catalyzed by the latter residues. A model for the anion transport mechanism in HR is suggested comprising the specific functions of channel-lining residues.     

Effects of the transport site conformation on the binding of external NAP-taurine to the human erythrocyte anion exchange system. Evidence for intrinsic asymmetry

External N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) inhibits human red cell chloride exchange by binding to a site that is distinct from the chloride transport site. Increases in the intracellular chloride concentration (at constant external chloride) cause an increase in the inhibitory potency of external NAP-taurine. This effect is not due to the changes in pH or membrane potential that usually accompany a chloride gradient, since even when these changes are reversed or eliminated the inhibitory potency remains high. According to the ping-pong model for anion exchange, such transmembrane effects of intracellular chloride on external NAP-taurine can be explained if NAP-taurine only binds to its site when the transport site is in the outward-facing (Eo or EClo ) form. Since NAP-taurine prevents the conformational change from EClo to ECli , it must lock the system in the outward-facing form. NAP-taurine can therefore be used just like the competitive inhibitor H2DIDS (4,4'-diisothiocyano-1,2- diphenylethane -2,2'-disulfonic acid) to monitor the fraction of transport sites that face outward. A quantitative analysis of the effects of chloride gradients on the inhibitory potency of NAP-taurine and H2DIDS reveals that the transport system is intrinsically asymmetric, such that when Cli = Clo, most of the unloaded transport sites face the cytoplasmic side of the membrane.     

Use of niflumic acid to determine the nature of the asymmetry of the human erythrocyte anion exchange system

Niflumic acid is a noncompetitive inhibitor of chloride exchange, which binds to a site different from the transport or modifier sites. When the internal Cl- concentration is raised, at constant extracellular Cl- , the inhibitory potency of niflumic acid increases. This effect cannot be attributed to changes in membrane potential, but rather it suggests that niflumic acid binds to the anion exchange protein band 3 only when the transport site faces outward. When the chloride gradient is reversed, with Clo greater than Cli , the inhibitory potency of niflumic acid decreases greatly, which indicates that the affinity of niflumic acid for band 3 with the transport site facing inward is almost 50 times less than when the transport site faces outward. Experiments in which Cli = Clo show no significant change in the inhibition by niflumic acid when Cl- is lowered from 150 to 10 mM. These data suggest that the intrinsic dissociation constants for Cl- at the two sides of the membrane are nearly equal. Thus, the chloride- loaded transport sites have an asymmetric orientation like that of the unloaded transport sites, with approximately 15 times more sites facing the inside than the outside. The asymmetry reflects an approximately 1.5 kcal/mol free energy difference between the inward-facing and outward-facing chloride-loaded forms of band 3. High concentrations of chloride (with Cli = Clo), which partially saturate the modifier site, have no effect on niflumic acid inhibition, which indicates that chloride binds equally well to the modifier site regardless of the orientation of the transport site.     

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.     

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.     

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.     

Biochemical characterization of halorhodopsin in native membranes

Procedures are described for selectively radiolabeling the protein moiety (haloopsin) or the chromophoric prosthestic group (retinal) of the light-driven chloride pump halorhodopsin in intact cells of Halobacterium halobium. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography, two retinal-binding polypeptides are observed to band near the known molecular weight of the halorhodopsin chromophoric polypeptide (25,000). Synthesis of one of these polypeptides is controlled by retinal and is sufficient for generation of complete halorhodopsin function. The other is constitutively produced by the cells and differs chemically from the haloopsin protein as indicated by differences in their V8 protease digestion patterns. V8 protease cleavage of haloopsin in its native membrane is compared with that of the protein in denaturing and nondenaturing detergents. Protease cleavage sites available in the denatured haloopsin molecule are hidden in its native membrane-integrated conformation and in nondenaturing detergent micelles. Treatment with a variety of proteases indicates susceptibility of a short terminal region of the haloopsin chain in its native conformation.     

Existence of two L photointermediates of halorhodopsin from Halobacterium salinarium, differing in their protein and water FTIR bands.

FTIR difference spectra were recorded for the photoreactions of halorhodopsin from Halobacterium salinarium at 170 and 250 K. Obvious differences at the two temperatures were noted in neither the visible spectra nor the FTIR bands of the chromophore. However, perturbation of Asp141 is observed in the L intermediate at 250 K but not at 170 K. We named these photoproducts La (at 170 K) and Lb (at 250 K). The spectrum of Lb is distinct from that of La also in the different shifts of water O-H stretching bands, and larger changes in the bands from the protein backbone with different sensitivities to varying the halide. These results suggest that the photocycle of halorhodopsin contains two L states, La and Lb, in which the structure of protein and internal water molecules is different but chloride stays at the same site close to the Schiff base.     

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.