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.     

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.     

Proton transport by bacteriorhodopsin through an interface film

Summary Interface films of purple membrane and lipid containing spectroscopically intact and oriented bacteriorhodopsin have been used as a model system to study the function of this protein. Small positive charges in surface potential (<1 mV) are detected upon illumination of these films at the air-water interface. These photopotentials, are not affected by overlaying the interface film with a thin layer (0.3 mm) of decane. However, they are dramatically increased when lipid soluble proton carriers FCCP or DNP are added to the decane. The polarity of the photopotential indicates that, in the light, positive charges are transported through the interface from the aqueous to the organic phase. The action spectrum of the photopotential is identical to the absorption spectrum of bacteriorhodopsin. Since bacteriorhodopsin molecules are oriented with their intracellular surface towards the aqueous subphase, the characteristics of the photopotential indicate that in the light bacteriorhodopsin translocates protons from its intracellular to its extracellular surface. The kinetics of the photopotential reveal that the rate and extent of proton transport are proportional both to the fraction of bacteriorhodopsin molecules excited and to the concentration of proton acceptor. The photopotentials result from changes in the ionic distribution across the decane-water interface and can be cancelled by lipid soluble anions.     

Kinetics of the light-driven proton movement in model membranes containing bacteriorhodopsin.

The short-circuit photoresponse of model membranes containing bacteriorhodopsin to short (35 ms) and long (3.5 s) light pulses is described. It is shown that if the light pulse is short compared with the charging and discharging times of the model membrane, the temporal response of the light-driven proton pump can be measured. Photoactive planar model membranes were formed both from biomolecular lipid membranes and from solid 6-micrometers thick Teflon septa coated with lipid and bacteriorhodopsin. The kinetic response of the pump is independent of the planar model membrane system in which it is incorporated. Experimental evidence indicates that the shape of the leading and trailing edges of the photoresponse curve for the pump deviates from simple exponential behavior. The short-circuit photoresponse of spinach chloroplast in a planar model membrane was also studied for comparison purposes.     

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.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

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.     

A measurement of the proton pump current generated by bacteriorhodopsin in black lipid membranes

The light-induced electrical current generated by black lipid membranes containing bacteriorhodopsin from Halobacterium halobium has been measured directly. It is shown that a measurement of membrane potential can also be used to obtain the proton pump current developed during illumination. Evidence is presented that the charge movement across the membrane is associated with the release of protons in the photoreaction cycle of bacteriorhodopsin. The time variation of the pump current when the light is turned on suggests the rapid depopulation of some initially occupied state.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

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.     

Lipidic cubic phase crystallization of bacteriorhodopsin and cryotrapping of intermediates: towards resolving a revolving photocycle

Bacteriorhodopsin is a small retinal protein found in the membrane of the halophilic bacterium Halobacterium salinarum, whose function is to pump protons across the cell membrane against an electrostatic potential, thus converting light into a proton-motive potential needed for the synthesis of ATP. Because of its relative simplicity, exceptional stability and the fundamental importance of vectorial proton pumping, bacteriorhodopsin has become one of the most important model systems in the field of bioenergetics. Recently, a novel methodology to obtain well-diffracting crystals of membrane proteins, utilizing membrane-like bicontinuous lipidic cubic phases, has been introduced, providing X-ray structures of bacteriorhodopsin and its photocycle intermediates at ever higher resolution. We describe this methodology, the new insights provided by the higher resolution ground state structures, and review the mechanistic implications of the structural intermediates reported to date. A detailed understanding of the mechanism of vectorial proton transport across the membrane is thus emerging, helping to elucidate a number of fundamental issues in bioenergetics.     

Blue light effect on proton pumping by bacteriorhodopsin.

Proton pumping in closed vesicular systems containing bacteriorhodopsin that is initiated by an orange flash, is diminished by a subsequent blue flash. This blue light effect is due to light absorbed by the photocycle intermediate M412 (M), which was formed by the orange flash. A kinetic analysis of the blue-light-induced reduction of proton pumping shows that of the two components of M, only the slowly decaying component is involved in the reduction of proton movement. This may be the first correlation between a proton movement and a specific photochemical intermediate of bacteriorhodopsin. Furthermore, we report that blue light, acting on the slowly decaying intermediate, probably causes a movement of the protons in a direction opposite to that normally seen for light absorbed by bacteriorhodopsin.     

Primary charge motions and light-energy transduction in bacteriorhodopsin

The bacteriorhodopsin protein (bR) in the cell membrane of Halobacterium halobium is a light driven proton pump. Many details are known about its structure and the molecular mechanism of proton translocation. The events may be characterized by: (1) the changes in light absorption after photon excitation (the photocycle); (2) the charge motion cycle inside the protein: the steps taken by the proton during translocation; (3) the retinal cycle. changes in isomerization and protonation; and (4) the opsin cycle: alterations of protonation of different amino acids in the apoprotein. From a review of existing data a more or less concise picture of the parallelism of the above four cycles emerges, which may be valuable as a model for understanding other types of molecular pumps.     

Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes

The expression of membrane proteins for functional and structural studies or medicinal applications is still not very well established. Membrane-spanning proteins that mediate the information flow of the extracellular side with the interior of the cell are prime targets for drug development methods that would allow screening techniques or high throughput formats are of particular interest. Here we describe a systematic approach to the liposome-assisted cell-free synthesis of functional membrane proteins. We demonstrate the synthesis of bacteriorhodopsin (bR(cf)) in presence of small unilamellar liposomes. The yield of bR(cf) per volume cell culture is comparable to that of bacteriorhodopsin in its native host. The functional analysis of bR(cf) was performed directly using the cell-free reaction mixture. Photocycle measurements reveal kinetic data similar to that determined for bR in Halobacterium salinarum cell-envelope vesicles. The liposomes can be attached directly to black lipid membranes (BLM), which allows measuring light activated photocurrents in situ. The results reveal a functional proton pump with properties identical to those established for the native protein.     

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.     

Moist and soft, dry and stiff: a review of neutron experiments on hydration-dynamics-activity relations in the purple membrane of Halobacterium salinarum

Twenty-five years of neutron experiments on hydration and thermal dynamics in purple membranes of Halobacterium salinarum are reviewed. Neutron diffraction, elastic and quasielastic scattering, allowed to map the distribution of water and lipids and to measure thermal fluctuations and correlation times in the membranes, under various conditions of temperature, hydration and lipid environment. Strong correlations were established between dynamics parameters and the activity of bacteriorhodopsin (the purple membrane protein), as a light driven proton pump supporting the hypothesis that the influence of hydration on activity is in fact due to its effects on membrane thermal dynamics. Hydrogen-deuterium labelling experiments highlighted stiffer and softer parts in the bacteriorhodopsin structure. The soft parts would allow the conformational changes involved in activity, while the stiffer ones may control a valve-like function in vectorial proton transfer.     

Transducer protein HtrI controls proton movements in sensory rhodopsin I

Sensory rhodopsin I (SR-I lambda(max) 587 nm) is a phototaxis receptor in the archaeon Halobacterium salinarium. Photoisomerization of retinal in SR-I generates a long-lived intermediate with lambda(max) 373 nm which transmits a signal to the membrane-bound transducer protein HtrI. Although SR-I is structurally similar to the electrogenic proton pump bacteriorhodopsin (BR), early studies showed its photoreactions do not pump protons, nor result in membrane hyperpolarization. These studies used functionally active SR-I, that is, SR-I complexed with its transducer HtrI. Using recombinant DNA methods we have expressed SR-I protein containing mutations in ionizable residues near the protonated Schiff base, and studied wild-type and site-specifically mutated SR-I in the presence and absence of the transducer protein. UV-Vis kinetic absorption spectroscopy, FT-IR, and pH and membrane potential probes reveal transducer-free SR-I photoreactions result in vectorial proton translocation across the membrane in the same direction as that of BR. This proton pumping is suppressed by interaction with transducer which diverts the proton movements into an electroneutral path. A key step in this diversion is that transducer interaction raises the pK(a) of the aspartyl residue in SR-I (Asp76) which corresponds to the primary proton-accepting residue in the BR pump (Asp85). In transducer-free SR-I, our evidence indicates the pK(a) of Asp76 is 7.2, and ionized Asp76 functions as the Schiff base proton acceptor in the SR-I pump. In the SR-I/HtrI complex, the pK(a) of Asp76 is 8.5, and therefore at physiological pH (7.4) Asp76 is neutral. Protonation changes on Asp76 are clearly not required for signaling since the SR-I mutants D76N and D76A are active in phototaxis. The latent proton-translocation potential of SR-I may reflect the evolution of the SR-I sensory signaling mechanism from the proton pumping mechanism of BR.     

Proteoliposomes with right-side-out oriented purple membrane/bacteriorhodopsin require cations inside for proton pumping

Proteoliposomes were prepared by sonication of phospholipids and blue membranes (cation-free purple membranes carrying little activity of light-driven proton pumping) in an acidic medium of very low ionic strength. The majority of the bacteriorhodopsin population in these proteoliposomes was in the right-side-out (as in living cells) orientation as judged from the resultant polypeptides after papain digestion. By raising the pH of sonication, the population of right-side-out oriented bacteriorhodopsin decreased, and consequently that of the inversely oriented one increased. In KCl and NaCl up to certain concentrations or in choline chloride even at high concentrations, in the light, the proteoliposomes with right-side-out bacteriorhodopsin did not pump protons, whereas those with inversely oriented bacteriorhodopsin did. The former began to pump only after cations were likely incorporated/permeated into the proteoliposome and reached the carboxyl terminal (cytosol) side of bacteriorhodopsin/purple membrane.     

菌紫质稳态异构体光致变色反应动力学初探

实验证实,在适当的酸度调节下暗适应菌紫质（BR）的光致变色反应由B→蓝膜→P→Q→B的循环转换构成。在无光照下,B、Q态在中性介质中,蓝膜、P态在酸性介质中均呈高化学稳定性;蓝膜→P和Q→B的态转换须分别用650nm和400nm可见光激励,用紫外－可见光谱对两个光化学过程的动力学特性进行监测,证实它们均为一级反应。菌紫质的四个稳态在可见光区具有不同的特征吸收波长,在信息记录方面可望有一定应用前景。     

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.