The photochemical reactions of bacterial sensory rhodopsin-I. Flash photolysis study in the one microsecond to eight second time window.

Halobacterium halobium Flx mutants are deficient in bacteriorhodopsin (bR) and halorhodopsin (hR). Such strains are phototactic and the light signal detectors are two additional retinal pigments, sensory rhodopsins I and II (sR-I and sR-II), which absorb maximally at 587 and 480 nm, respectively. A retinal-deficient Flx mutant, Flx5R, overproduces sR-I-opsin and does not show any photochemical activity other than that of sR-I after the pigment is regenerated by addition of all-trans retinal. Using native membrane vesicles from this strain, we have resolved a new photointermediate in the sR-I photocycle between the early bathointermediate S610 and the later intermediate S373. The new form, S560, resembles the L intermediate of bR in its position in the photoreaction cycle, its relatively low extinction, and its moderate blue shift. It forms with a half-time of approximately 90 microseconds at 21 degrees C, concomitant with the decay of S610. Its decay with a half-time of 270 microseconds parallels the appearance of S373. From a data set consisting of laser flash-induced absorbance changes (300 ns, 580-nm excitation) measured at 24 wavelengths from 340 to 720 nm in a time window spanning 1 microsecond to 8 s we have calculated the spectra of the photocycle intermediates assuming a unidirectional, unbranched reaction scheme.     

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.     

Circular dichroism of halorhodopsin: comparison with bacteriorhodopsin and sensory rhodopsin I

Circular dichroic (CD) spectra of three related protein pigments from Halobacterium halobium, halorhodopsin (HR), bacteriorhodopsin (BR), and sensory rhodopsin I (SR-I), are compared. In native membranes the two light-driven ion pumps, HR and BR, exhibit bilobe circular dichroism spectra characteristic of exciton splitting in the region of retinal absorption, while the phototaxis receptor, SR-I, exhibits a single positive band centered at the SR-I absorbance maximum. This indicates specific aggregation of protein monomers of HR, as previously noted [Sugiyama, Y., & Mukohata, Y. (1984) J. Biochem. (Tokyo) 96, 413-420], similar to the well-characterized retinal/retinal exciton interaction in the purple membrane. The absence of this interaction in SR-I indicates SR-I is present in the native membrane as monomers or that interactions between the retinal chromophores are weak due to chromophore orientation or separation. Solubilization of HR and BR with nondenaturing detergents eliminates the exciton coupling, and the resulting CD spectra share similar features in all spectral regions from 250 to 700 nm. Schiff-base deprotonation of both BR and HR yields positive CD bands near 410 nm and shows similar fine structure in both pigments. Removal of detergent restores the HR native spectrum. HR differs from BR in that circular dichroic bands corresponding to both amino acid and retinal environments are much more sensitive to external salt concentration and pH. A theoretical analysis of the exciton spectra of HR and BR that provides a range of interchromophore distances and orientations is performed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light-driven primary sodium ion transport in halobacterium halobium membranes

Light-induced sodium extrusion from H halobium cell envelope vesicles proceeds largely through an uncoupler-sensitive pathway involving bacteriorhodopsin and a proton/sodium antiporter. Vesicles from bacteriorhodopsin-negative strains also extrude sodium ions during illumination, but this transport is not sensitive to uncouplers and has been proposed to involve a light-energized primary sodium pump. Proton uptake in such vesicles is passive, and under steady-state illumination the large electrical potential (negative inside) is just balanced by a pH difference (acid inside), so that the protonmotive force is near zero. Action spectra indicated that this effect of illumination is attributable to a pigment absorbing near 585 nm (of 568 for bacteriorhodopsin). Bleaching of the vesicles by prolonged illumination with hydroxylamine results in inactivation of the transport; retinal addition causes partial return of the activity. Retinal addition also causes the appearance of an absorption peak at 588 nm, while the absorption of free retinal decreases. The 588 nm pigment is present in very small quantities (0.13 nmole/mg protein), and behaves differently from bacteriorhodopsin in a number of respects. Vesicles can be prepared from bacteriorhodopsin-containing H halobium strains in which primary transport for both protons and sodium can be observed. Both pumps appear to cause the outward transport of the cations. The observations indicate the existence of a second retinal protein, in addition to bacteriorhodopsin, in H halobium, which is associated with primary sodium translocation. The initial proton uptake normally observed during illumination of whole H halobium cells may therefore be a passive flux in response to the primary sodium extrusion.     

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.     

Photoactive retinal pigments in haloalkaliphilic bacteria

Light-induced fast transient absorbance changes were detected by time-resolved spectroscopy in 38 of 51 haloalkaliphilic isolates from alkaline salt lakes in Kenya and the Wadi Natrun in Egypt. They indicate the presence of two retinal pigments, Pf and Ps, which undergo cyclic photoreactions with half-times of 2 ms and 500 ms respectively. Pf absorbs maximally near 580 nm and Ps near 500 nm. The pigments differ in their sensitivity to hydroxylamine and detergent bleaching and the photoreactions of Pf are strongly dependent on chloride concentration. Of the 38 pigment-containing strains, 29 possess both Pf and Ps, 9 possess only Ps. Inhibition of retinal synthesis with nicotine blocks pigment formation and addition of retinal restores it. Hydroxylamine-bleached pigments can be reconstituted with retinal or retinal analogues. Their similarity to the retinal pigments of Halobacterium halobium strongly suggests that they are also rhodopsin-like retinyledene proteins. Pf in all properties tested is almost identical to halorhodopsin, the light-driven chloride pump of H. halobium, and may serve the same function in the haloalkaliphiles. Ps has photocycle kinetics similar to sensory rhodopsin and a far-blue-shifted long-lived photocycle intermediate, but its ground state absorption maximum is near 500 nm instead of 587 nm. We have not found a bacteriorhodopsin-like pigment in the haloalkaliphiles.     

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.     

The photochemical cycle of bacteriorhodopsin.

The reaction cycle of bacteriorhodopsin in the purple membrane isolated from Halobacterium halobium has been studied by optical absorption spectroscopy using low-temperature and flash kinetic techniques. After absorption of light, bacteriohodopsin passes through at least five distinct intermediates. The temperature and pH dependence of the absorbance changes suggests that branch points and/or reversible steps exist in this cycle. Flash spectroscopy in the presence of a pH-indicating dye shows that the transient release of a proton accompanies the photoreaction cycle. The proton release occurs from the exterior and the uptake is on the cytoplasmic side of the membrane, as required by the function of bacteriorhodopsin as a light-driven proton pump. Proton translocating steps connecting release and uptake are indicated by deuterium isotope effects on the kinetics of the cycle. The rapid decay of a light-induced linear dichroism shows that a chromophore orientation change occurs during the reaction cycle.     

Evidence that the long-lifetime photointermediate of s-rhodopsin is a receptor for negative phototaxis in Halobacterium halobium

The effect of blue background light on behavioral response of Halobacterium halobium to step-like stimulation with green-orange attractant light was examined. The results strongly support the previously proposed hypothesis that a long-lifetime photointermediate of s-rhodopsin is the photoreceptor for repellent light: the step-like increase in green-orange light was convertible from attractant stimulus to repellent one, when the cells were constantly illuminated with blue light. No difference of the threshold intensity of the blue background light was observed between the mutant strain that lacks both bacteriorhodopsin and halorhodopsin and the wild type strain, suggesting that the two light-driven ion pumps are not participant in sensing attractant light.     

Effect of salt on photocycle and ion-pumping of halorhodopsin and third rhodopsinlike pigment of Halobacterium halobium

The cytoplasmic membranes of Halobacterium halobium contain at least three retinal pigments: bacteriorhodopsin (bR), halorhodopsin (hR), and a third rhodopsinlike pigment (tR). The amplitudes of the phototransient in the photolysis of hR and tR were measured in various salt solutions. Halogen ion (except fluoride) was required to retain the photocycle of hR. Parallels between the amplitude of the phototransient of hR and the magnitude of the photo-induced tetraphenylphosphonium (TPP+) uptake suggests that hR is a light-driven halogen pump, which supports the hypothesis by Schobert and Lanyi (J. Biol. Chem., 1982, 257:10306-10313). The order of effectiveness of halogen was Br- greater than Cl- greater than I-. On the other hand, no specific ion was required to retain the photocycle of tR, and tR was concluded to be nonelectrogenic.     

Characterization of the chromophore of the third rhodopsin-like pigment of Halobacterium halobium and its photoproduct.

Halobacterium halobium contains at least three retinal-containing pigments: bacteriorhodopsin, halorhodopsin, and a third rhodopsin-like pigment (tR) absorbing at approximately 590 nm, tR590. Illumination of tR590 gives rise to a very long-lived blue absorbing photoproduct, tR370. Using high-performance liquid chromatography we show that the chromophore of tR590 is primarily all-trans retinal and its conversion by light to tR370 causes the chromophore to isomerize primarily to the 13-cis conformation. Irradiation of the tR370 gives rise to a transient photoproduct absorbing at approximately 520 nm that decays back to the initial pigment tR590. In addition to all-trans retinal, the apomembrane of tR can also combine with 13-cis retinal but not with the 9- or 11-cis isomers.     

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.     

Transient proton inflows during illumination of anaerobic Halobacterium halobium cells

In Halobacterium halobium strain R1 containing both bacteriorhodopsin (bR) and halorhodopsin (hR), the light-driven proton uptake has been experimentally resolved into three transient inflows which are superimposed on the larger proton outflow. Under anaerobic conditions the early proton uptake consists of two components: (i) an inflow which can be blocked using the ATPase inhibitor, Dio-9, and (ii) an inflow which can be abolished by low concentrations (less than 125 nM) of triphenyltin chloride (TPT) with no inhibition of ATP synthesis. At pH 6 these two inflows are approximately equal in magnitude and duration. Measurements of buffering capacity and internal pH indicate that Dio-9 does not alter the passive proton-hydroxyl permeability of the cell membrane and that TPT at these low concentrations slightly decreases it. At later times of illumination (iii) another transient light-driven proton inflow occurs. This inflow is most evident during the first illumination after cells have been stored for extended times in the dark. The internal potassium concentration is not changed by storage, but apparently sodium is taken up, and we attribute the third inflow to sodium extrusion in exchange for protons. These results demonstrate the existence of three distinct triggered secondary proton inflows through the cell membrane. The proton inflow, which can be inhibited by Dio-9, correlates with proton-dependent ATP synthesis. The second inflow, which disappears in the presence of low TPT concentrations, is a passive proton uptake through an otherwise unidentified channel in response to electrogenic chloride pumping by bacteriorhodopsin and/or halorhodopsin. The third system correlates with the Na+/H+ antiporter function that has been demonstrated in H. halobium cell envelope vesicles. In contrast to observations on hR-containing vesicles, which can develop substantial Cl- gradients, the electroneutral OH-/Cl- exchange function can be demonstrated in intact cells only at TPT concentrations greater than 500 nM.     

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.     

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.     

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.     

Membrane potential modulates photocycling rates of bacterial rhodopsins

Effects of membrane potential on photochemical reactions of three retinal-containing chromoproteins in Halobacterium halobium, sensory rhodopsin I (sR-I), bacteriorhodopsin, and halorhodopsin, are described. Each of the three exhibits a decreased rate of thermal decay of its principal intermediate when photoactivated in an artificially energized compared to a deenergized membrane. The similar response of the three pigments suggests a voltage-dependent conformational change common to their respective photocycles. Spectral and kinetic properties of the sR-I photochemical reaction cycle were measured in phototactic H. halobium cells, and differences from in vitro photocycle kinetics were attributable to the electrical membrane potential present in vivo. In vivo sR-I photocycling rates were reproduced in envelope vesicle preparations in the presence of a valinomycin-induced potassium diffusion potential.     

Iso-halorhodopsin: a stable, 9-cis retinal containing photoproduct of halorhodopsin.

Dark-adapted halorhodopsin is a mixture of 13-cis and all-trans retinal chromophoric species. It is known that illumination with blue light increases the all-trans content, and this is reversed partially by brief red illumination. We now find that extended red-light illumination produces a third spectroscopic form. Analysis of composite absorption spectra recorded during various illumination regimes yielded the spectrum for the new species, whose absorption is shifted approximately 100 nm to the blue. The isomeric composition of retinal extracted from the illuminated pigment indicates that this form contains 9-cis retinal. This species, which we name iso-halorhodopsin, is stable in the dark at room temperature for at least a day, but can be quantitatively reconverted into a mixture of all-trans and 13-cis halorhodopsin by blue-light illumination. A kinetic scheme for the isomeric interconversions was drawn up, where iso-halorhodopsin is produced from either all-trans halorhodopsin only, or both 13-cis and all-trans forms. This kind of scheme is supported by the finding that red illumination of halo-opsin reconstituted with 13-trans-locked retinal will generate iso-halorhodopsin. A similar experiment with 13-cis-locked retinal could not be done because reconstitution with this retinal analogue was not possible. The photoreaction that leads to iso-halorhodopsin can be readily demonstrated in detergent-solubilized halorhodopsin or in halorhodopsin in liposomes made from phosphatidylcholine plus phosphatidyl-ethanolamine, but only to much reduced extent in cell envelope vesicles and in halorhodopsin incorporated into liposomes made from halobacterial polar lipids.     

Time-resolved FT-IR spectroscopic investigation of the pH-dependent proton transfer reactions in the E194Q mutant of bacteriorhodopsin

The photoreaction of the E194Q mutant of bacteriorhodopsin has been investigated at various pH values by time-resolved step-scan Fourier-transform infrared difference spectroscopy employing the attenuated total reflection technique. The difference spectrum at pH 8.4 is comparable to the N-BR difference spectra of the wild type with the remarkable exception that D85 is deprotonated. Since the retinal configuration is not perturbed by the E194Q mutation, it is concluded that there is no interaction of D85 with retinal during the lifetime of the N state. At pH 6, a consecutive state to the O intermediate is detected in which D212 is transiently protonated. The comparison with wild-type bacteriorhodopsin reveals that protonation of D212 represents an intermediate step during proton transfer from D85 to the proton release group in the final stage of the reaction cycle. The described effects are more pronounced in the E194Q mutant than in the E204Q mutant demonstrating different roles of these two glutamates/glutamic acids at least in the final stages of the catalytic cycle of bacteriorhodopsin.