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.     

Analogies between halorhodopsin and bacteriorhodopsin

The light-activated proton-pumping bacteriorhodopsin and chloride ion-pumping halorhodopsin are compared. They belong to the family of retinal proteins, with 25% amino acid sequence homology. Both proteins have seven alpha helices across the membrane, surrounding the retinal binding pocket. Photoexcitation of all-trans retinal leads to ion transporting photocycles, which exhibit great similarities in the two proteins, despite the differences in the ion transported. The spectra of the K, L, N and O intermediates, calculated using time-resolved spectroscopic measurements, are very similar in both proteins. The absorption kinetic measurements reveal that the chloride ion transporting photocycle of halorhodopsin does not have intermediate M characteristic for deprotonated Schiff base, and intermediate L dominates the process. Energetically the photocycle of bacteriorhodopsin is driven mostly by the decrease of the entropic energy, while the photocycle of halorhodopsin is enthalpy-driven. The ion transporting steps were characterized by the electrogenicity of the intermediates, calculated from the photoinduced transient electric signal measurements. The function of both proteins could be described with the 'local access' model developed for bacteriorhodopsin. In the framework of this model it is easy to understand how bacteriorhodopsin can be converted into a chloride pump, and halorhodopsin into a proton pump, by changing the ion specificity with added ions or site-directed mutagenesis.     

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.     

Characterization of the proton-transporting photocycle of pharaonis halorhodopsin

The photocycle of pharaonis halorhodopsin was investigated in the presence of 100 mM NaN(3) and 1 M Na(2)SO(4). Recent observations established that the replacement of the chloride ion with azide transforms the photocycle from a chloride-transporting one into a proton-transporting one. Kinetic analysis proves that the photocycle is very similar to that of bacteriorhodopsin. After K and L, intermediate M appears, which is missing from the chloride-transporting photocycle. In this intermediate the retinal Schiff base deprotonates. The rise of M in halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multiphasic. Intermediate N cannot be detected, but a large amount of O accumulates. The multiphasic character of the last step of the photocycle could be explained by the existence of a HR' state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes its sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride photocycle of halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchanged by exchanging chloride with azide.     

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.     

Conserved amino acids in F-helix of bacteriorhodopsin form part of a retinal binding pocket

A 3-dimensional model for the retinal binding pocket in the light-driven proton pump, bacteriorhodopsin, is proposed on the basis of spectroscopic studies of bacteriorhodopsin mutants. In this model Trp-182, Pro-186 and Trp-189 surround the polyene chain while Tyr-185 is positioned close to the retinylidene Schiff base. This model is supported by sequence homologies in the F-helices of bacteriorhodopsin and the related retinal proteins, halorhodopsin and rhodopsins.     

Trans-cis isomerization of retinal and a mechanism for ion translocation in halorhodopsin

The chromophore in halorhodopsin (HR) which acts as a light-driven chloride pump in halobacteria shares many properties with its counterpart in bacteriorhodopsin (BR): (i) a similar retinal protein interaction, (ii) trans to cis isomerization and (iii) similar intermediates of its photocycle. One major difference between the two chromoproteins is that the HR chromophore does not become deprotonated during its photocycle. A mechanism for the photocycle of HR is presented, which, in close analogy to an earlier proposed mechanism for BR, involves the sequence of all-trans 13-cis, 14s-cis 13-cis all-trans isomerizations of the chromophore, a Schiff base of retinal. In contrast to the situation in BR the 13-cis, 14s-cis13-cis isomerization is induced not by deprotonation of the retinal Schiff base chromophore but rather by the movement of an anion (Cl^-) towards the protonated nitrogen of the Schiff's base. The suggested mechanism involves the Schiff base directly in the chloride translocation in halorhodopsin.     

Secondary structure of halorhodopsin

Ultraviolet circular dichroism (CD) spectroscopy in the interval from 190 to 240 nm has been used to estimate the secondary structural content of halorhodopsin (hR), a light-driven chloride pump isolated from the membranes of Halobacterium halobium. Least-squares curve fitting of the CD spectrum for hR solubilized with octyl glucoside yields an alpha-helical content of approximately 50% and a beta-structure content of approximately 30%. The CD spectrum of hR is unaffected by the absence or presence of chloride ions or by the ionic strength of the medium. The CD spectrum of halorhodopsin is very similar to that of bacteriorhodopsin, indicating that these light-driven pumps possess nearly identical fractions of alpha- and beta-secondary structures.     

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.     

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.     

Spectroscopic discrimination of the three rhodopsinlike pigments in Halobacterium halobium membranes.

Membranes of Halobacterium halobium contain two photochemically reactive retinal pigments in addition to the proton pump bacteriorhodopsin. One, halorhodopsin, is also an electrogenic ion pump with a fast (on a scale of milliseconds) photoreaction cycle. The other, s-rhodopsin, is active in the same spectral region, but has a much slower photoreaction cycle (on a scale of seconds). S-rhodopsin is not an electrogenic ion pump and its properties suggest it functions as the receptor pigment for phototaxis. All three pigments have very similar absorption spectra. The recent isolation of mutants deficient in both bacteriorhodopsin and halorhodopsin and in retinal synthesis has allowed us to resolve the absorption spectra of s-rhodopsin and halorhodopsin. At neutral pH s-rhodopsin has an absorption maximum at 587 +/- 2 nm and halorhodopsin at 578 +/- 2 nm. At pH 10.8, lambda max for s-rhodopsin is shifted to 552 nm and extinction decreases slightly (15%) while halorhodopsin loses all extinction above 500 nm. Both effects are fully reversible and allow determination of the amounts of s-rhodopsin and halorhodopsin in membrane preparations containing both pigments. Both pigments were present in earlier studies of H. halobium membranes, and in view of these findings, several observations must be reinterpreted.     

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.     

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.     

Isomeric composition of retinal chromophore in dark-adapted bacteriorhodopsin

1. Retinal isomers extracted from the acid-hydrolysate of cetyltrimethylammonium bromide-treated dark-adapted bacteriorhodopsin (bRD) were analyzed in a high performance liquid chromatograph (HPLC) system. The extract from bRD contains almost equal molar amounts of both 13-cis retinal and all-trans retinal isomers. The extent of isomerization and the yield of both isomers during the isolation process were investigated by the application of the same extraction procedure to artificial bacteriorhodopsin reconstituted with 13-cis retinal isomer (13-cis bacteriorhodopsin) and also to light-adapted bacteriorhodopsin (bRL) which has been shown to contain only the all-trans isomer (all-trans bacteriorhodopsin). 2. A reconstituted bacteriorhodopsin, which had been prepared from apo-bacteriorhodopsin and an equimolar mixture of both 13-cis retinal and all-trans retinal isomers, showed an absorption spectrum having the same maximum wavelength as that of bRD even at the beginning of the reconstitution process. 3. Analysis of the photosteady states of bRD at -190 degrees C revealed that it was composed of two different species, one having 13-cis retinal and the other having all-trans retinal isomers in approximately equal molar amounts. These two also gave their respective photoproducts. 4. From these results it can be concluded that bRD contains both 13-cis retinal and all-trans retinal isomers in nearly equal molar amounts as its chromophore.     

Light and dark adaptation of halorhodopsin

Dark incubation of envelope vesicles derived from a strain of Halobacterium halobium that lacks bacteriorhodopsin but contains halorhodopsin and a third rhodopsin-like pigment caused a decrease in the flash yield [the amplitude of a transient absorbance change of flash reactive component(s) by flash] of halorhodopsin but not the rhodopsin-like pigment. The flash yield decreased to reach a low steady level after incubation for about 4 days in the dark. The flash yield of halorhodopsin at any stage of dark incubation was increased by actinic illumination of the vesicles. The flash yield at 490 nm (absorbance increase) was found to be approximately proportional to that at 590 nm (absorbance decrease). These results indicate that halorhodopsin in the envelope vesicles has two forms, dark and light adapted, and that the halorhodopsin phototransient absorbing at 490 nm is originated from the light-adapted form. A difference spectrum between these two forms of halorhodopsin shows that the light-adapted halorhodopsin was red-shifted from the dark-adapted form. The light-induced membrane potential was measured by tetraphenylphosphonium uptake. The uptake by the dark-adapted vesicles was slower than that by the light-adapted vesicles, suggesting that only the light-adapted halorhodopsin has ion-transporting activity.     

Nature of the principal photointermediate of halorhodopsin

Two alternative hypotheses have been presented as to the nature of the principal halorhodopsin photointermediate: a) it is a form whose its absorption band is shifted from the 575 nm position to 500 or 520 nm, and b) it is a form whose absorption band is shifted to only about 565 nm, but with an altered band shape so it exhibits a fortuitous difference peak near 500 nm. Such a shift with a maximum near 500 nm is also obtained in the dark when chloride is removed from the sample, suggesting the hypothesis that the spectral changes reflect the transient detachment of chloride from a binding site (Ogurusu et al, J. Biochem. Tokyo 95, 1073-1082, 1984). Comparison of the quantum yields of flash-induced absorption changes in halorhodopsin and bacteriorhodopsin strongly suggests, however, that hypothesis b) is untenable.     

Isolation and characterization of halorhodopsin from Halobacterium halobium

Chromoprotein of a light-driven chloride pump, halorhodopsin (HR), was isolated from Halobacterium halobium L-33, which contains HR and "slowly cycling rhodopsin-like pigment" (SR) but lacks bacteriorhodopsin (BR). The isolation was run in the presence of more than 2 M NaCl, which was required to preserve this halophilic retinal protein. Cell envelope vesicles were washed with Tween-20 to remove 80% of the proteins. The residual membranes were solubilized with 0.5% C12E9, which had little effect on the photochemical activities of HR and SR. HR was purified by passing it through a hydroxyapatite and then a phenyl-Sepharose column in 2 M NaCl and 0.5% C12E9. The absorption maximum of HR was 578 nm and the ratio of absorbance at 280 nm to 580 nm was 1.52. The apparent molecular weight of HR was 20,000 on polyacrylamide gel electrophoresis in the presence of SDS. The characteristic, bilobed CD spectrum of HR in the visible region suggested that HR exists as an oligomer in both its membrane-bound and isolated forms.     

Why 11-cis-Retinal?

The C₂₀ diterpenoid compound retinal is the chromophore of thevisual pigments the rhodopsins, and the pigments present inHalobacterium halobium, namely, bacteriorhodopsin (proton pump),halorhodopsin (chloride pump), and the sensory rhodopsins (phototaxisreceptor). In all cases, they are bound covalently to the receptorprotein by a protonated Schiff base. However, in rhodopsins,the retinal is the 11-cis isomer, whereas in H. halobium pigmentsit is the all-trans isomer. Why did Nature choose retinal asthe chromophore, and why 11-cis in some cases and all-transin other cases? Also why is the chromophore a protonated Schiffbase? These points are addressed after giving an outline ofthe current status of the various photoreceptor pigments     

Retinal-Protein Interactions in Halorhodopsin from Natronomonas pharaonis: Binding and Retinal Thermal Isomerization Catalysis

Halorhodopsin from Natronomonas pharaonis (NpHR) is a member of the retinal protein group and serves as a light-driven chloride pump in which chloride ions are transported through the membrane following light absorption by the retinal chromophore. In this study, we examined two main issues: (1) factors controlling the binding of the retinal chromophore to the NpHR opsin and (2) the ability of the NpHR opsin to catalyze the thermal isomerization of retinal isomers. We have revealed that the reconstitution process of pharaonis HR (NpHR) pigment from its apoprotein and all-trans retinal depends on the pH, and the process has a pK_a of 5.8 ± 0.1. It was proposed that this pK_a is associated with the pK_a of the lysine residue that binds the retinal chromophore (Lys256). The pigment formation is regulated by the concentration of sodium chloride, and the maximum yield was observed at 3.7 M NaCl. The low yield of pigment in a lower concentration of NaCl (< 3 M) may be due to an altered conformation adopted by the apomembrane, which is not capable of forming the pigment. Unexpectedly and unlike the apomembrane of bacteriorhodopsin, NpHR opsin produces pigments with 11-cis retinal and 9-cis retinal owing to the thermal isomerization of these retinal isomers to all-trans retinal. The isomerization rate depends on the pH, and it is faster at a higher pH. The pK_a value of the isomerization process is similar to the pK_a of the binding process of these retinals, which suggests that Lys256 is also involved in the isomerization process. The isomerization is independent of the sodium chloride concentration. However, in the absence of sodium chloride, the apoprotein adopts such a conformation, which does not prevent the isomerization of retinal, but it prevents a covalent bond formation with the lysine residue. The rate and the thermodynamic parameter analysis of the retinal isomerization by NpHR apoprotein led to the conclusion that the apomembrane catalyzes the isomerization via a triplet mechanism.