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.     

The enthalpy changes upon hydrolysis of guanosine triphosphate anhydride and ester bonds

The effects of N,N′-dicyclohexylcarbodiimide (DCCD), triphenyltin chloride (TPT), and 3,5-di-tert-butyl-4-hydroxybenzylidenemalonomtrile (SP6847) were tested on the light-dependent activities of Halobacterium halobium R₁mR which contains a new retinal protein pigment designated as halorhodopsin but no bacteriorhodospin. DCCD inhibited ATP synthesis either in the light- or in the dark-aerobic conditions without affecting the light-induced proton uptake (ΔH⁺). Although DCCD lowered the membrane potential under dark-anaerobic conditions, the potential increased in the light as high as the control (the light-dependent membrane potential increment Δψ became apparently larger in the presence of DCCD). TPT had negligible effect on ATP synthesis both in the dark or in the light but inhibited markedly ΔH⁺ and partly Δψ. After R₁mR was treated with DCCD, TPT abolished ΔH⁺ almost completely but Δψ only partly. The remaining Δψ was collapsed by SF6847 with a concomitant proton incorporation (pH increase). These results led to the following postulations: (i) In R₁mR, ATP is synthesized by a H⁺-ATPase coupled either to respiration and/or light energization by halorhodopsin; (ii) the majority of protons are incorporated in the light by a mechanism which differs from H⁺-ATPase but is driven by the Δψ generated by halorhodopsin; (iii) TPT acts in this system as a chloride/hydroxide exchanger; (iv) the uncoupler SF6847 carries protons into cells in response to Δψ.     

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.     

ESR studies of light-dependent volume changes in cell envelope vesicles from Halobacterium halobium

Volume changes in illuminated cell envelope vesicles, prepared from various Halobacterium halobium strains, were measured with an ESR method. We demonstrated light-dependent swelling of vesicles which contained halorhodopsin (an inward-directed light-driven chloride pump), and shrinking of vesicles which contained bacteriorhodopsin (an outward-directed light-driven proton pump coupled to a proton/sodium antiporter). The swelling of the halorhodopsin vesicles was not inhibited by uncouplers or gramicidin, but the shrinking of the bacteriorhodopsin-vesicles was abolished by these ionophores. These findings confirm earlier models for ion circulation in these systems. Vesicles from strains which contained both pigments showed relatively small net volume changes upon illumination. A scheme of ionic transport in H. halobium cells is suggested, in which the inward movement of K⁺ exceeds the outward movement of Na⁺, and the difference equals the Cl⁻ uptake, so as to provide the net gain of KCl necessary for volume increases during cell growth.     

Suggestion of existence of two forms of halorhodospin in alkaline solution

Illumination of halorhodopsin (hR590) with orange light in alkaline solution produced a 410 nm absorbing species (hR410), which returned to hR590 upon blue light illumination. The amount of the flash-reactive species of hR590 was estimated by the flash-yield. Illumination with orange light decreased the flash yield, due to the formation of hR410. Blue light illumination of this sample led to the increase of the yield, which was larger than that before orange light illumination. In dark, the yield decreased gradually in 3-4 days. The scheme is proposed in which there exist two forms of hR590.     

Determination of membrane potential with lipophilic cations. Comparison of estimated values with various phosphonium ions

The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined. The lipophilic ions used constitute a homologous series of (Phe)₃-P⁺-(CH₂)_n-CH₃ (n = 0–5) and tetraphenylphosphonium (TPP⁺). In the absence of membrane potential, the binding of probes to the membrane was measured. For the probes of n = 0 and n = 1, and for TPP⁺, binding followed the Langmuir adsorption isotherm. For other probes, analysis revealed the presence of two, high- and low-affinity, binding sites. Upon illumination, which generated the membrane potential, the probe molecules were accumulated into the vesicles. If we ignore the membrane-potential-dependent binding of the probe molecules, the estimated values are larger when the probe used is more hydrophobic. We have tested some models describing the amount of probe bound on membranes in terms of concentration of free probe inside and outside the vesicles. No model has fulfilled the criterion of valid estimation that the membrane potentials estimated are independent of probes used. An experimental method for the estimation of true membrane potential is proposed. Effects of tetraphenylboron on the estimation of membrane potential and on the transport rate of phosphonium cations were examined.     

Sidedness of membrane structures in Rhodopseudomonas sphaeroides. Electrochemical titration of the spectrum changes of carotenoid in spheroplasts,spheroplast membrane vesicles and chromatophores

The shift of the carotenoid absorption spectrum induced by illumination and valinomycin-K⁺ addition was investigated in membrane structures with different characteristics and opposite sidednesses isolated from Rhodopseudomonas sphaeroides. Right-side-out membrane structures were prepared by isotonic lysozyme-EDTA treatment of the cells (spheroplasts) and by hypotonic treatment of spheroplasts (spheroplast membrane vesicles). Inside-out membrane structures (“chromatophores”) were obtained by treating spheroplast membrane vesicles by French press or sonication.The membrane structures with either sidedness showed the same light-induced change of the “red shift” type. However, the absorbance change by K⁺ addition in the presence of valinomycin in the right-side-out membrane structures were opposite to that in the inverted vesicles, “blue shift” in the former and “red shift” in the latter. The carotenoid absorbance change was linear to membrane potential, calculated from the concentration of KCl added, with a reference on the cytoplasmic side, through positive and negative ranges.     

Photophosphorylation in cell envelope vesicles from Halobacteriumhalobium

We have prepared vesicles from cell envelope membranes of $\text{Halobacterium}\text{halobium}$ strains R₁ and ET-15 which are able to synthesize ATP in response to illumination. This photophosphorylation is inhibited by dicyclohexylcarbodiimide (DCCD) and by phloretin. ATP synthesis in L vesicles from the R₁ strain (which contain bacteriorhodopsin) is inhibited by the protonophore 1799 but not by valinomycin. In M vesicles from the R₁ strain and in ET-15 vesicles (both contain halorhodopsin) photophosphorylation is inhibited by both 1799 and valinomycin. These data are consistent with the idea that light-driven ATP synthesis can be coupled to the electrochemical H⁺ gradient generated by bacteriorhodopsin or by halorhodopsin through the membrane potential component of protonmotive force.     

Photochromism of halorhodopsin. cis/trans isomerization of the retinal around the 13-14 double bond

The isomeric composition of retinal in membrane-bound and in purified but detergent-free, dark-adapted halorhodopsin was found to be about 70% 13-cis and 30% all-trans. Any illumination increased the all-trans content relative to the dark-adapted state, but blue illumination shifted the isomeric composition more toward all-trans while red illumination of blue-adapted samples shifted it more toward 13-cis. In the presence of chloride this photoisomerization caused the kind of photochromic behavior reported earlier in Smith, S. O., Marvin, M. J., Bogomolni, R. A., and Mathies, R. A. (1984) J. Biol. Chem. 259, 12326-12329, i.e. blue light caused the absorption maximum to move toward longer wavelengths and red light reversed the shift. Only the all-trans chromophore exhibited the complete photocycle described earlier in detergent-solubilized halorhodopsin, and this was the form that could be associated with light-driven chloride transport activity in cell envelope vesicles. In the absence of chloride the spectroscopic changes caused by illumination were much smaller. Reconstitution of bleached preparations with 13-cis- and all-trans-retinal, in the presence and absence of chloride, confirmed that the difference between the absorption maxima of the two isomeric forms of the chromophore is affected by chloride: 13-cis-halorhodopsin absorbs at about 567-568 nm with and without chloride, and the all-trans pigment absorbs near 568 nm in the absence of chloride, but at 578 nm in its presence. The simplest explanation of this finding is that most of the red-shift which accompanies the 13-cis----all-trans transition originates from electrostatic interaction of the retinal with chloride bound in its vicinity.     

Carotenoid transformations underlying the blue absorbance change in flashed leaves during the induction of oxygen evolution

The blue absorbance change occurring in flashed bean (Phaseolus vulgaris L.) leaves when exposed to continuous light (first observed by Strasser; Strasser, R.J. (1973) Arch. Int. Physiol. Biochem. 81, 935–955) is caused by the conversion of the following xanthophylls: violaxanthine → antheraxanthine → zeaxanthine. This conclusion is derived from the simultaneous occurrence of both reactions: (a) In flashed leaves, blue absorbance change and xanthophyll conversion take place under strong (2 mW · cm^?2) but not under weak (0.02 mW · cm^?2) white light. (b) In chloroplasts isolated from flashed leaves, the blue absorbance change occurs in the dark under conditions that also induce the xanthophyll conversion. (c) Blue absorbance change and xanthophyll conversion are both inhibited by dithiothreitol. In addition, the light-induced blue absorbance change is reversed in the dark if aerobic conditions are maintained, i.e. under conditions that in normal leaves favor the reversal of the above reaction sequence.The significance of the xanthophyll conversion is discussed in relation to other phenomena occurring in flashed leaves after exposure to continuous illumination.     

Studies on cephalopod rhodopsin. Conformational changes in chromophore and protein during the photoregeneration process

The ultraviolet absorbance of squid and octopus rhodopsin changes reversibly at 234 nm and near 280 nm in the interconversion of rhodopsin and metarhodopsin. The absorbance change near 280 nm is ascribed to both protein and chromophore parts. Rhodopsin is photoregenerated from metarhodopsin via an intermediate, P380, on irradiation with yellow light (λ > 520 nm). The ultraviolet absorbance decreases in the change from rhodopsin to metarhodopsin and recovers in two steps; mostly in the process from metarhodopsin to P380 and to a lesser extent in the process from P380 to rhodopsin. P380 has a circular dichroism (CD) band at 380 nm and its magnitude is the same order as that of rhodopsin. Thus it is considered that the molecular structure of P380 is close to that of rhodopsin and that the chromophore is fixed to opsin as in rhodopsin. In the change from metarhodopsin to P380, the chromophore is isomerized from the all-trans to the 11-cis form, and the conformation of opsin changes to fit 11-cis retinal. In the change from P380 to rhodopsin, a small change in the conformation of the protein part and the protonation of the Schiff base, the primary retinal-opsin link, occur.     

Phototactic behaviour in Aphidius gifuensis (Hymenoptera:Braconidae)

We investigated the spectral sensitivity and response to light intensity of Aphidius gifuensis (Hymenoptera: Braconidae), a key natural enemy of the green peach aphid, Myzus persicae (Hemiptera: Aphididae). We used 15 monochromatic lights (emitting various specific wavelengths from 340 to 689 nm) and white light. Monochromatic light of different wavelengths and white light elicited photopositive behaviour from A. gifuensis. The strongest response was stimulated by blue light (492 nm), which induced a movement of 43.5 cm, a response that differed from all other groups. This was followed by green light (568 nm) and UV-light (380 nm). There was no significant response to orange light (601 nm) or red light (649, 668 and 689 nm) from A. gifuensis. The response intensity curve for A. gifuensis to monochromatic light (492 nm) decreased as light intensity increased. At 568 nm, the phototactic response showed an ‘S’ shaped curve. But at 628 nm, the phototactic response rose continuously with increasing intensity. We report here that the visual system of A. gifuensis is composed of three spectrum receptors, attuned to UV, blue and green light. While light intensity is a key factor in determining the photopositive response of A. gifuensis, the effect of intensity varies by wavelength.     

An H-ATPase Assay: Proton Pumping and ATPase Activity Determined Simultaneously in the Same Sample

A continuous spectrophotometric assay of H⁺-ATPase activity was developed by combining two well-known methods for measuring proton pumping and ATPase activity. Proton uptake into plasma membrane vesicles from Avena sativa L. (cv Rhiannon) was monitored as the absorbance decrease at 495 nm of the ΔpH probe acridine orange. Simultaneously, ATPase activity was measured by following the absorbance decrease at 340 nanometers by coupling ATP hydrolysis enzymatically to the oxidation of NADH. This H⁺-ATPase assay is convenient for determining the relative relationship between ATP hydrolysis and proton pumping.     

The ATP-dependent generation of membrane potential by sub-bacterial vesicles from the marine bacterium,Vibrio alginolyticus

Addition of ATP leads to the accumulation of the permeant anion PCB⁻ by sub-bacterial vesicles from Vibrio alginolyticus. This accumulation is caused by Δψ generation by ATPase, the effect being inhibited by CCCP, gramicidin D and DCCD. Δψ values may be increased by incubation of sub-bacterial vesicles at room temperature and with the protein fraction isolated according to Beechey et al. [(1975) Biochem. J. 148, 533–537] from another portion of the sub-bacterial vesicles. Δψ generation is observable only in the presence of Mg²⁺ at high concentrations (optimum ≈ 30 mM). Proceeding from experimental data we assume that Mg²⁺ reduces passive H⁺ conductivity of the vesicle membranes. Thus, a Δψ-generating ATPase has been shown for the first time in V. alginolyticus membranes.     

Photoreduction and Oxidation of Cytochrome f in Bundle Sheath Cells of Maize

The photo-oxidation of cytochrome f (cytochrome c₅₅₄) in bundle sheath cells isolated from leaves of maize (Zea mays var. DS 606A) has been compared with that in intact maize leaf and in isolated pea leaf cells (Pisum sativum L.). In all cases, illumination with red light caused a negative absorbance change at 554 nm which was attributed to the oxidation of cytochrome f. The extent of this change was greater using monochromatic red light at wavelengths above 700 nm compared with wavelengths below 700 nm. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea abolished this difference in bundle sheath cells. After illumination for 1 minute or longer in bundle sheath cells, reduction of cytochrome f in the dark was rapid only if the wavelength of the illuminating light was below 700 nm. In the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethlyurea, reduction was slow after illumination at all wavelengths.     

Phototoxic Action Spectrum on a Retinal Pigment Epithelium Model of Age-Related Macular Degeneration Exposed to Sunlight Normalized Conditions

Among the identified risk factors of age-related macular degeneration, sunlight is known to induce cumulative damage to the retina. A photosensitive derivative of the visual pigment, N-retinylidene-N-retinylethanolamine (A2E), may be involved in this phototoxicity. The high energy visible light between 380 nm and 500 nm (blue light) is incriminated. Our aim was to define the most toxic wavelengths in the blue-green range on an in vitro model of the disease. Primary cultures of porcine retinal pigment epithelium cells were incubated for 6 hours with different A2E concentrations and exposed for 18 hours to 10 nm illumination bands centered from 380 to 520 nm in 10 nm increments. Light irradiances were normalized with respect to the natural sunlight reaching the retina. Six hours after light exposure, cell viability, necrosis and apoptosis were assessed using the Apotox-Glo Triplex™ assay. Retinal pigment epithelium cells incubated with A2E displayed fluorescent bodies within the cytoplasm. Their absorption and emission spectra were similar to those of A2E. Exposure to 10 nm illumination bands induced a loss in cell viability with a dose dependence upon A2E concentrations. Irrespective of A2E concentration, the loss of cell viability was maximal for wavelengths from 415 to 455 nm. Cell viability decrease was correlated to an increase in cell apoptosis indicated by caspase-3/7 activities in the same spectral range. No light-elicited necrosis was measured as compared to control cells maintained in darkness. Our results defined the precise spectrum of light retinal toxicity in physiological irradiance conditions on an in vitro model of age-related macular degeneration. Surprisingly, a narrow bandwidth in blue light generated the greatest phototoxic risk to retinal pigment epithelium cells. This phototoxic spectrum may be advantageously valued in designing selective photoprotection ophthalmic filters, without disrupting essential visual and non-visual functions of the eye.     

Photodynamic Alteration of Sodium Currents in Lobster Axons

Photodynamic alteration of lobster giant axons drastically changed the magnitude and kinetics of sodium currents seen under voltage clamp using the sucrose gap technique. Illumination of axons following treatment with acridine orange or eosin Y decreased the maximum sodium conductance to a zero asymptote as an exponential function of illumination time. Normal sodium inactivation was slowed, with τ_h more than doubled depending on experimental conditions. A second slower inactivation rate developed occasionally. τ_h was altered little, if at all. Sodium current "tails" were not prolonged. At maximum light intensity and with eosin Y as sensitizer leakage current increased after 4–10 sec in light. These changes were irreversible. Decreases in maximum sodium conductance correlated highly with increases in time to peak sodium current. The magnitude of change varied linearly with light intensity. The action spectra for eosin Y and acridine orange peaked near 545 and 505 nm, respectively. The magnitude of change varied with preillumination dye exposure time in a quasi-exponential approach to a maximum effect. Sodium dithionite protected the axon from photodynamic change.     

A unifying concept for ion translocation by retinal proteins

First, halorhodopsin is capable of pumping protons after illumination with greenand blue light in the same direction as chloride. Second, mutated bacteriorhodopsin where the proton acceptor Asp85 and the proton donor Asp96 are replaced by Asn showed proton pump activity after illumination with blue light in the same direction as wildtype after green light illumination. These results can be explained by and are discussed in light of our new hypothesis: structural changes in either molecule lead to a change in ion affinity and accessibility for determining the vectoriality of the transport through the two proteins.     

Intercellular power transmission along trichomes of cyanobacteria

An attempt at demonstrating lateral power transmission over millimeter distances along a coupling membrane has been undertaken. Trichomes of the multicellular filamentous cyanobacteria Phormidium uncinatum were illuminated with a very narrow light beam forming a light spot that covered only 4–5% of a 1–2 mm long cyanobacterial trichome. Such illumination was found to support motility (gliding along agar surface) of the trichome under conditions when the light was the only energy source. It was also shown that illumination with the light spot caused rotation of rings of slime (accompanying the operation of the ‘motors’ responsible for the motility of cyanobacteria) not only in the illuminated, but also in the distal, nonilluminated part of the trichome. Electric potential transmission along trichomes was revealed by means of the extracellular electrode technique. The light spot was found to induce generation of an electric potential difference between two electrodes in the dark region of the trichomes, which were placed at different distances from the illuminated end. Cutting the trichomes between the light spot and the closest ‘dark’ electrode abolished this effect. Valinomycin + K⁺ and carbonyl cyanide p-trifluoromethoxyphenylhydrazone affected the potential difference formation between two ‘dark’ electrodes much stronger than that between a light and a dark electrode. All the light spot-induced effects develop in the seconds time scale. Both the amplitudes and the kinetics of the potential difference measured with four electrodes placed along the trichome prove to be in good agreement with the theoretical curves computed on the basis of the electric cable equation. It is concluded that transcellular power transmission in the form of Δψ takes place along trichomes of cyanobacteria. This confirms the hypothesis about the biological function of Δψ as a transportable form of energy.     

Action spectra for C-glucosylflavone accumulation in Hordeum vulgare plumules

Five-day-old etiolated barley plumules contain the C-glucosylflavones saponarin, lutonarin, and lutonarin 3′-methyl ether. When harvested 24 hr after illumination, increased flavonoid levels were essentially linear with increased energies of monochromatic light at seven wavelengths between 450 and 750 nm. Action spectra for saponarin and for a mixture of lutonarin and its 3′-methyl ether were determined between 380 and 760 nm at 6.6 kerg·cm^?2. The saponarin action spectrum showed distinct peaks at 620 and at 660 nm. These two peaks were similar in their photoreversibility when followed by either 6·6 or 34 kerg·cm^?2 of far-red light. Phytochrome is apparently the photoreceptor for the saponarin action spectrum. Lutonarin and its 3′-methyl ether showed peaks at 520 580, 620 and near 660 nm. The 660 nm peak was not photoreversible by 6·6 kerg·cm^?1, but was by 34 kerg·cm_?2, of far-red light. Phytochrome and protochlorophyll are the likely photoreceptors for these 3′-substituted flavonoids.