Reconstitution of Biological Molecular generators of electric current. Bacteriorhodopsin.

1. Photoinduced generation of electric current by bacteriorhodopsin, incorporated into the planar phospholipid membrane, has been directly measured with conventional electrometer techniques. 2. Two methods for bacteriorhodopsin incorporation have been developed: (a) formation of planar membrane from a mixture of decane solution of phospholipids and of the fraction of violet fragments of the Halobacterium halobium membrane (bacteriorhodopsin sheets), and (b) adhesion of bacteriorhodopsin-containing reconstituted spherical membranes (proteoliposomes) to the planar membrane in the presence of Ca2+ or some other cations. In both cases, illumination was found to induce electric current generation directed across the planar membrane, an effect which was measured by macroelectrodes immersed into electrolyte solutions on both sides of the membrane. 3. The maximal values of the transmembrane electric potential were of about 150 mV at a current of about 10(-11) A. The electromotive force measured by means of counterbalancing the photoeffect by an external battery, was found to reach the value of 300 mV. 4. The action spectrum of the photoeffect coincides with the bacteriorhodopsin absorption spectrum (maximum about 570 nm). 5. Both components of the electrochemical potential of H+ ions (electric potential and delta pH) across the planar membrane affect the bacteriorhodopsin photoelectric response in a fashion which could be expected if bacteriorhodopsin were a light-dependent electrogenic proton pump. 6. La3+ ions were shown to inhibit operation of those bacteriorhodopsin which pump out H+ ions from the La3+-containing compartment. 7. The photoeffect, mediated by proteoliposomes associated with thick planar membrane, is decreased by gramicidin A at concentrations which do not influence the planar membrane resistance in the light. On the contrary, a protonophorous uncoupler, trichlorocarbonylcyanidephenylhydrazone, decreases the photoeffect only if it is added at a concentration lowering the light resistance. The dark resistance is shown to be higher than the light one, and decreases to the light level by gramicidin. 8. A simple equivalent electric scheme consistent with the above results has been proposed.     

Mechanism of generation and regulation of photopotential by bacteriorhodopsin in bimolecular lipid membrane. The quenching effect of blue light

Photoelectric properties of bacteriorhodopsin incorporated into a bimolecular lipid membrane were investigated with special regard to the mechanism of photoelectric field generation. It was shown that besides its proton pump and electric generator functions bacteriorhodopsin works as a possible molecular regulator of the light-induced membrane potential. When a bimolecular lipid membrane containing bacteriorhodopsin is continuously illuminated in its main visible absorption band, and afterwards by superimposed blue light matching the absorption band of the long-living photobleached bacteriorhodopsin (M₄₁₂) as well, the latter either enhances or decreases the steady-state photoresponse, depending upon the intensity of the green light. Thus, the additional blue-light illumination tends to cause the resultant photoelectric membrane potential to become stabilized. Two alternative schemes are tentatively proposed for the photochemical cycle of bacteriorhodopsin whereby blue light can control photovoltage generation. A kinetic model of the proton pump and the regulation of the photoelectric membrane potential is presented. This model fits all the experimental findings, even quantitatively. From the model some kinetic and physical parameters of this light-driven pump could be determined.     

Mechanism of generation and regulation of photopotential by bacteriorhodopsin in bimolecular lipid membrane.

Photoelectric properties of bacteriorhodopsin incorporated into a bimolecular lipid membrane were investigated with special regard to the mechanism of photoelectric field generation. It was shown that besides its proton pump and electric generator functions bacteriorhodopsin works as a possible molecular regulator of the light-induced membrane potential. When a bimolecular lipid membrane containing bacteriorhodopsin is continuously illuminated in its main visible absorption band, and afterwards by superimposed blue light matching the absorption band of the long-living photobleached bacteriorhodopsin (M412) as well, the latter either enhances or decreases the steady-state photoresponse, depending upon the intensity of the green light. Thus, the additional blue-light illumination tends to cause the resultant photoelectric membrane potential to become stabilized. Two alternative schemes are tentatively proposed for the photochemical cycle of bacteriorhodopsin whereby blue light can control photovoltage generation. A kinetic model of the proton pump and the regulation of the photoelectric membrane potential is presented. This model fits all the experimental findings, even quantitatively. From the model some kinetic and physical parameters of this light-driven pump could be determined.     

The effect of trypsin treatment on the incorporation and energy-transducing properties of bacteriorhodopsin in liposomes

Bacteriorhodopsin and trypsin-modified bacteriorhodopsin have been reconstituted into liposomes by means of a low pH-sonication procedure. The incorporation of bacteriorhodopsin in these proteoliposomes is predominantly in the same direction as in vivo and the direction of proton pumping is from inside to outside the liposomes. The direction of proton translocation and electrical potential generation was studied as a function of the reconstitution pH. Light-dependent proton extrusion and generation of a Δp, interior negative and alkaline was observed at a reconstitution pH below 3.0 using bacteriorhodopsin, and at a pH below 3.5 using trypsin-modified bacteriorhodopsin. The shift in inflection point is explained in terms of differences between bacteriorhodopsin and trypsin-modified bacteriorhodopsin in a specific protein-phospholipid interaction which depends on the surface charge density of the cytoplasmic side of bacteriorhodopsin. The magnitude of the protonmotive force (Δp) generated by trypsin-modified bacteriorhodopsin in liposomes was quantitated. Illumination of the proteoliposomes resulted in the generation of a high Δp (135 mV, inside negative and alkaline), with a major contribution of the pH gradient. The ionophores nigericin and valinomycin induced, respectively, a compensatory interconversion of ΔpH into Δψ and vice versa. If no endogenous proton permeability of the membrane would exist, a protonmotive force could be generated of − 143 mV as electrical potential alone or − 162 mV as pH gradient alone.     

Bacteriorhodopsin-mediated photoelectric responses in lipid/water systems

Summary Bacteriorhodopsin-mediated photopotential generation has been studied in two kinds of lipid/water systems: (1) decane solution of asolectin was used as the lipid phase; (2) a mixture of bacteriorhodopsin sheets and hexane solution of phosphatidyl choline was applied onto a water surface to form a monolayer, and then the monolayer was covered with a 0.3-mm decane layer. In both cases, illumination was found to induce formation of an electric potential difference, with the bulk water phase being found negative when measured with a vibrating electrode. In the latter, but not in the former, system small amounts of a protonophorous uncoupler were found to stimulate the photoresponse. Large amounts of the uncoupler proved depressing in both systems. Phenyldicarbaundecaborane anion (PCB^–) was shown to substitute for the uncoupler, being much more potent both as an activator and as an inhibitor of the photoresponse. In both studied systems, gramicidin A inhibits the photoresponse, the effect being greatly potentiated by K⁺, Na⁺ or H⁺ ions.In the system decane solution of asolectin/water, an Ag/AgCl electrode immersed into the lipid phase can be used instead of a vibrating electrode. All the measured features of the photoelectric responses observed with any of these electrodes were found to be quite similar to those inherent in a phospholipid-impregnated collodion film adsorbing bacteriorhodopsin sheets on one of its surfaces.A scheme is discussed built on the assumption that photopotentials in all the studied systems are due to an uphill light-dependent transport of H⁺ ions from the bulk water phase to a water cavity localized between a bacteriorhodopsin sheet and the surface of the bulk lipid phase. Thus, the above lipid/water systems containing bacteriorhodopsin are composed of four, rather than two, phases, as was supposed previously.Bacteriorhodopsin-mediated photopotential generation has been studied also in the decane/water system without phospholipids. This system with bacteriorhodopsin sheets added to the water phase demonstrates a light-dependent photoelectric response reaching 1.5 V, which can be measured only by a vibrating electrode. The photoresponse starts after a lag period of several seconds. Switching off the light results in the reversal of the light-induced electric potential change. The off-effect also has a lag period. The action spectrum of the photoresponse shows at least two maxima: a smaller at 560 nm and a larger at <420 nm. Free retinal can substitute for bacteriorhodopsin in the studied system. All the above effects disappear if, instead of air, argon is used. In the system decane solution of asolectin/water, a slow photoelectric response of this type can be demonstrated at neutral pH in the presence of gramicidin and at pH 4 without gramicidin. A suggestion is put forward that the slow photoelectric response is due to an interface Volta-potential change induced by a product of photooxidation of bacteriorhodopsin and/or free retinal released from bacteriorhodopsin.     

Temporal characteristics of bacteriorhodopsin as a molecular biological generator of current

Generation of electric potential difference by bacteriorhodopsin proteoliposomes incorporated into the phospholipid-impregnated collodion film has been studied. It is shown that illumination of this film by continuous light gives rise to the generation of an electric potential difference across the film (plus on the bacteriorhodopsin-free side), which can be as high as 300 mV. Short unsaturating flash inducing single turn-over of bacteriorhodopsin generates the potential difference which is a function of the flash intensity (70 mV at 3 mjoule light). The flash-induced photoelectric response consists of four phases. (1) Very fast (tau less than 1 microsec) generation of a potential difference (minus in the bacteriorhodopsin-free compartment). The amplitude of this phase is rather small (1--5 mV). (2) Fast phase of positive charging of the bacteriorhodopsin-free compartment (tau = 25--50 microsec). (3) Slow phase of positive charging of this compartment (tau = 6--12 msec) Amplitude of the second phase is to that of the third as 1 : 2. (4) A very slow phase of discharge of the flash-induced potential difference (tau = 1 sec at 10(8) ohm X cm2 film resistance). The third phase was specifically inhibited by La3+. Both the second and the third phases are decelerated by substitution of D2O in 4.5--5 and 2 times, respectively, while the amplitude of the first phase increases. Prolonged storage of the system in the dark (tua = 20--25 min) causes the decrease in the amplitudes of the second and the third phases as if the amount of active bacteriorhodopsin molecules were increased by factor 2. Such an inhibition was reversed by 30--60 sec illumination of the system. The dark adaptation is accompanied by some increase in the first phase amplitude. Comparison of these data with results of other studies on bacteriorhodopsin suggests that (1) the first phase is due to the photoinduced change in the retinal dipole; (2) the second phase corresponds to H+ transfer from Schiff base to the water solution in the proteoliposome interior; 3) the third phase represents H+ transfer from the incubation mixture to Schiff base; (4) the dark adaptation is a result of transition of photoelectrochemically active all-trans-retinal to the inactive 13-cis-retinal.     

pH对菌紫质分子的旋转运动和光电响应的影响

用闪光诱导瞬间二向色性方法测量了不同pH条件下的菌紫质分子在脂质囊泡中的旋转扩散运动.在人工平扳膜(BLM)系统中测量了不同pH条件下菌紫质分子的光电响应.在pH3至8.3的范围内没有明显观察到菌紫质分子在膜中旋转运动上的差别.pH低于3时,菌紫质分子旋转运动受到影响;pH高于11时,观察不到旋转扩散运动.在BLM系统中测量了pH2到pH11范围内菌紫质分子的光电响应信号,随着pH的增加,无论紫膜碎片还是单体菌紫质分子的光电响应逐渐由照光后快速正信号并快速衰减及撤光时的快速负信号并逐渐衰减变成慢的正信号.pH高于9.4时,单体菌紫质分子的光电响应信号由正变负,pH高于11时,观察不到信号.     

delta psi-mediated signalling in the bacteriorhodopsin-dependent photoresponse.

It has been shown previously that the proton-pumping activity of bacteriorhodopsin from Halobacterium salinarium can transmit an attractant signal to the bacterial flagella upon an increase in light intensity over a wide range of wavelengths. Here, we studied the effect of blue light on phototactic responses by the mutant strain Pho8l-B4, which lacks both sensory rhodopsins but has the ability to synthesize bacteriorhodopsin. Under conditions in which bacteriorhodopsin was largely accumulated as the M412 bacteriorhodopsin photocycle intermediate, halobacterial cells responded to blue light as a repellent. This response was pronounced when the membrane electric potential level was high in the presence of arginine, active oxygen consumption, or high-background long-wavelength light intensity but was inhibited by an uncoupler of oxidative phosphorylation (carbonyl cyanide 3-chlorophenylhydrazone) and was inverted in a background of low long-wavelength light intensity. The response to changes in the intensity of blue light under high background light was asymmetric, since removal of blue light did not produce an expected suppression of reversals. Addition of ammonium acetate, which is known to reduce the pH gradient changes across the membrane, did not inhibit the repellent effect of blue light, while the discharge of the membrane electric potential by tetraphenylphosphonium ions inhibited this sensory reaction. We conclude that the primary signal from bacteriorhodopsin to the sensory pathway involves changes in membrane potential.     

Investigation by focused laser beam scanning of the photoelectric activity of bacteriorhodopsin-containing lipid bilayers.

The photoelectric activity of different parts of lipid bilayer containing bacteriorhodopsin was investigated by moving a small actinic light spot across the Plateau-Gibbs border and the bimolecular part of this reconstituted model membrane. The results give direct evidence that bacteriorhodopsin incorporated into the bimolecular region of the lipid membrane is responsible for the photoelectric activity of this system. A technique for scanning the photoelectric activity of a modified bimolecular lipid membrane is described in detail.     

Photoelectric properties of a detector based on dried bacteriorhodopsin film

The photoelectric response of a detector using dried bacteriorhodopsin (bR) film as the light sensing material is mathematically modeled and experimentally verified in this paper. The photocycle and proton transfer kinetics of dried bR film differ dramatically from the more commonly studied aqueous bR material because of the dehydration process. The photoelectric response of the dried film is generated by charge displacement and recombination instead of transferring a proton from the cytoplasmic side to the extracellular side of the cell membrane. In this work, the wild-type bR samples are electrophoretically deposited onto an indium tin oxide (ITO) electrode to construct a simple multiple layered photo-detector with high sensitivity to small changes in incident illumination. The light absorption characteristics of the thin bR film are mathematically represented using the kinetics of the bR photocycle and the charge displacement theorem. An electrically equivalent RC circuit is used to describe the intrinsic photoelectric properties of the film and external measurement circuitry to analyze the detector's response characteristics. Simulated studies and experimental results show that the resistance of the dried bR film is in the order of 10(11) Omega. When the input impedance of the measurement circuitry is one order of magnitude smaller than the dried film, the detector exhibits a strong differential response to the original time-varying light signal. An analytical solution of the equivalent circuit also reveals that the resistance and capacitance values exhibited by the dried bR film, in the absence of incident light, are almost twice as large as the values obtained while the material is under direct illumination. Experimental observations and a predictive model both support the notion that dried bR film can be used in simple highly sensitive photo-detector designs.     

Photocurrent response of bacteriorhodopsin adsorbed on bimolecular lipid membranes

The photo response of bacteriorhodopsin adsorbed on a bimolecular lipid membrane has been investigated using short-circuit current measurements. The results revealed a biphasic current vs. time curve for the photocurrent at pH values of approx. 7. This phenomenon could be modified by altering either the value of the external applied electrical field or the proton concentration differences.The observed effects of the external applied voltage, pH gradient and lipophilic proton carriers enabled us to conclude that the bacteriorhodopsin can be adsorbed in two different states, which give rise to a pumping effect and a flux of protons in opposite directions.A theoretical analysis of the photocycle in relation to the electrical field which acts on the proton uptake and release is proposed. The main effect of this field is to diminish the pumping rate due to the proton motive force resulting from the creation of space-charge in the vicinity of purple membrane fragments.     

Bacteriorhodopsin-mediated photophosphorylation in Halobacterium halobium.

The rate of halobacterial photophosphorylation was found to be a linear function of light intensity over a wide range (between 1 and 20 mW/cm2). At higher light intensities (above 25 mW/cm2) the ATP-synthesizing system itself limits the maximal rate of photophosphorylation. The optimal external pH range for this type of photophosphorylation is between pH 6.2 and 7.2 external. The photophosphorylation rate is directly proportional to the bacteriorhodopsin content of the cells. The quantum requirement for photophosphorylation was found to be 22 +/- 5 photons per ATP molecule synthesized. According to Mitchell's chemiosmotic hypothesis of energy coupling phosphorylation can be driven by a membrane potential or a pH gradient or a combination of both. From the results of experiments with drugs which abolish or reduce either one of the two components we conclude that the major driving force for photophosphorylation above an external pH value of 6.5 is the membrane potential, while at more acidic pH value the pH gradient becomes dominating. We did not observe a correlation between a transient alkalinization of the medium and ATP-synthesis upon illumination under certain conditions.     

Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure.

The difference Fourier transform infrared spectrum for the N intermediate in the photoreaction of the light-adapted form of bacteriorhodopsin can be recorded at pH 10 at 274 K (Pfefferlé, J.-M., Maeda, A., Sasaki, J., and Yoshizawa, T. (1991) Biochemistry 30, 6548-6556). Under these conditions, Asp96-->Asn bacteriorhodopsin gives a photoproduct which shows changes in protein structure similar to those observed in N of wild-type bacteriorhodopsin. However, decreased intensity of the chromophore bands and the single absorbance maximum at about 400 nm indicate that the Schiff base is unprotonated, as in the M intermediate. This photoproduct was named MN. At pH 7, where the supply of proton is not as restricted as at pH 10, Asp96-->Asn bacteriorhodopsin yields N with a protonated Schiff base. The Asn96 residue, which cannot deprotonate as Asp96 in wild-type bacteriorhodopsin, is perturbed upon formation of both MN at pH 10 and N at pH 7. We suggest that the reprotonation of the Schiff base is preceded by a large change in the protein structure including perturbation of the residue at position 96.     

Energy-linked nicotinamide-nucleotide transhydrogenase. Light-driven transhydrogenase catalyzed by transhydrogenase from beef heart mitochondria reconstituted with bacteriorhodopsin

Purified nicotinamide-nucleotide transhydrogenase from beef heart mitochondria was co-reconstituted with bacteriorhodopsin to from transhydrogenase-bacteriorhodopsin vesicles that catalyze a 20-fold light-dependent and uncoupler-sensitive stimulation of the reduction of NADP+ and NADP+ analogs by NADH and a 50-fold shift of the nicotinamide nucleotide ratio. In the presence of light, the transhydrogenase-bacteriorhodopsin vesicles catalyzed a pronounced light intensity-dependent inward proton pumping as indicated by a pH shift of the medium. As indicated by pH shifts, proton pumping by the bacteriorhodopsin essentially paralleled the light-driven transhydrogenase. Addition of valinomycin increased the pH shift twice with a concomitant 50% inhibition of the light-driven transhydrogenase, whereas nigericin inhibited the pH shift completely and the light-driven transhydrogenase partially. Taken together, these results suggest that transhydrogenase and bacteriorhodopsin interact through a delocalized proton-motive force. Possible partial reactions of transhydrogenase were investigated with transhydrogenase-bacteriorhodopsin vesicles energized by light. Reduction of oxidized 3-acetylpyridine adenine dinucleotide by NADH, previously claimed to represent partial reactions, was found to require NADPH. Similarly, reduction of thio-NADP+ by NADPH required NADH. It is concluded that these reactions do not represent partial reactions.     

Modeling the membrane potential generation of bacteriorhodopsin

The archaeon Halobacterium salinarum can grow phototrophically with only light as its energy source. It uses the retinal containing and light-driven proton pump bacteriorhodopsin to enhance the membrane potential which drives the ATP synthase. Therefore, a model of the membrane potential generation of bacteriorhodopsin is of central importance to the development of a mathematical model of the bioenergetics of H. salinarum. To measure the current produced by bacteriorhodopsin at different light intensities and clamped voltages, we expressed the gene in Xenopus laevis oocytes. We present current-voltage measurements and a mathematical model of the current-voltage relationship of bacteriorhodopsin and its generation of the membrane potential. The model consists of three intermediate states, the BR, L, and M states, and comparisons between model predictions and experimental data show that the L to M reaction must be inhibited by the membrane potential. The model is not able to fit the current-voltage measurements when only the M to BR phase is membrane potential dependent, while it is able to do so when either only the L to M reaction or both reactions (L to M and M to BR) are membrane potential dependent. We also show that a decay term is necessary for modeling the rate of change of the membrane potential.     

The pH-dependence of photochemical intermediates of O and P in bacteriorhodopsin by continuous light

The pH-dependence of the O and P intermediates in the photocycle of bacteriorhodopsin (bR) on the intensity and duration of the exciting flash was investigated for bR glycerol suspensions and bR gelatin films. Green and red laser flashes (532 and 670 nm) were utilized to generate a photoequilibrium state of bR and O at ambient temperature, and UV-vis spectroscopy was used to determine the photoconversion for the bR suspensions and films. The maximal concentration of the O intermediate was observed to be pH-dependent and the dependency was most pronounced at a slightly alkaline pH values. The photochemical conversion from the O to P intermediate was investigated for both bR suspensions and films. The P intermediate was only found in bR gelatin film. These results indicate that bR gelatin film may be an attractive candidate for the information storage based on P intermediate. It is possible, with red light, to create photoproducts which are thermally stable at ambient temperature and that can be photochemically erased.     

The photocycle of bacteriorhodopsin immobilized in poly(vinyl alcohol) film

The kinetics of photoelectric and optical signals were measured on samples containing oriented purple membranes immobilized in a poly(vinyl alcohol) film and on purple membranes introduced into a PVA-H2O mixture. The bacteriorhodopsin photocycle in the PVA-H2O mixture was complete. The only observed changes were the slowing down of the optical and electrical signals in relation to the M412-O640 and O640-bRall-trans steps. In the PVA film the O640 intermediate disappeared and a negative photoelectric signal appeared.     

The proton pump bacteriorhodopsin is a photoreceptor for signal transduction in Halobacterium halobium.

Halobacterium halobium swims by rotating its polarly inserted flagellar bundle. The cells are attracted by green-to-orange light which they can use for photophosphorylation but flee damaging blue or ultraviolet light. It is generally believed that this kind of 'colour vision' is achieved by the combined action of two photoreceptor proteins, sensory rhodopsins-I and -II, that switch in the light the rotational sense of the bundle and in consequence the swimming direction of a cell. By expressing the bacteriorhodopsin gene in a photoreceptor-negative background we have now demonstrated the existence of a proton-motive force sensor (protometer) and the function of bacteriorhodopsin as an additional photoreceptor covering the high intensity range. When the bacteriorhodopsin-generated proton-motive force drops caused by a sudden decrease in light intensity, the cells respond by reversing their swimming direction. This response does not occur when the proton-motive force is saturated by respiration or fermentation.     

pH-dependence of interaction between melittin and bacteriorhodopsin

Melittin differentially slowed down the fast (M412f) and the slow (M412s) decay components of the photocyde intermediate M of trimeric bacteriorhodopsin in purple membrane while it accelerated the M412s of Triton X-100-solubilized bacteriorhodopsin monomers. Raising the bulk pH could enhance the effect of melittin on the M412s of bacteriorhodopsin in these two states. From pH 5.5 to 8.8, melittin slightly influenced the yield of intermediate M in purple membrane, whereas the yield of M412s decreased and subsequently reversed with the addition of melittin. Moreover, the monomeric bacteriorhodopsin bleached more readily in the presence of melittin and the higher pH made the bleaching effect of melittin more intensive as well. These results re-certify our former suggestions that there was electrostatic interaction between melittin and bacteriorhodopsin, and indicate that the biphasic M decay may not result from the well-known linear kinetic scheme (M→N →BR). At last the mechanisms underlying the interact