Order of proton uptake and release by bacteriorhodopsin at low pH.

The order of proton uptake and release in an aqueous suspension of purple membrane in response to a light flash has been investigated at lowered pH. pH indicator dyes and a flash spectrophotometer were used for the study. At pH 6.6 it was found that the release of protons from the purple membrane precedes uptake, as reported by other investigators. At pH 5.9, 4.9, and 4.1 it was also found that release precedes uptake. These results are not in agreement with those of previous investigators.     

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side.

In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21 °C.

In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds.

Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds.

The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.     

Aggregation and proton release of purple and white membranes following cleavage of the carboxyl-terminal tail of bacteriorhodopsin

Our results indicate that the previously reported decrease in proton release by proteolyzed purple membrane sheets was due merely to the aggregation state of these preparations and not to the loss of the carboxyl-terminal tail. Changes in H+/M412 ratios obtained for purple and white membrane preparations correlate with the measured aggregation. White membrane preparations consistently exhibit H+/M412 ratios more than twice those measured for native purple membranes under the same conditions. Quasi-elastic light scattering was used to characterize the size of isolated purple and white membrane sheets before and after proteolysis. The results clearly show that native purple membrane preparations are larger in size than would be expected and that, following trypsin treatment, they are on average more than an order of magnitude larger. Negative staining electron microscopy showed that the purple membrane became aggregated in stacked arrays. Bleaching and reconstitution with retinal also affect aggregation, but iodination or nitration of purple membrane does not affect the measured size. The average size of white membranes is smaller; this is consistent with results of electron microscopy and the size increase is much less than that of purple membranes following trypsin treatment. No size change occurs with retinal reconstitution. In aggregated purple membrane preparations, protons and other cations are unable to exchange freely with the aqueous medium, explaining why proteolysis lowers the proton release from purple membrane sheets in suspension.     

Actinic light-energy dependence of proton release from bacteriorhodopsin

Measuring the light-density (fluence) dependence of proton release from flash excited bacteriorhodopsin with two independent methods we found that the lifetime of proton release increases and the proton pumping activity, defined as a number of protons per number of photocycle, decreases with increasing fluence. An interpretation of these results, based on bending of purple membrane and electrical interaction among the proton release groups of bacteriorhodopsin trimer, is presented.     

Light-induced currents from oriented purple membrane: II. Proton and cation contributions to the photocurrent

The sign of B2, the micro-second component of the photocurrent from oriented purple membrane, is that of positive charge moving away from the purple membrane in the direction of proton release. B2 could be due to internal dipole or proton movement, proton release, or metal cation release. We found that the waveform of B2 is virtually insensitive to changes in the salt concentration as long as it is >40 mM KCl, >5 mM CaCl₂, or >0.5 mM LaCl₃. However, below these limits, B2's apparent rate of decay increases as the salt concentration decreases without any change in the initial amplitude. This salt dependence suggests that B2 is due to a positive charge, either a metal cation or a proton, moving from the membrane into the solution. That the positive charge is not a metal cation is suggested by the waveform of B2 remaining unchanged upon replacing the cations both in solution and in the binding sites of the purple membrane. Direct evidence that the positive charge movement is due to protons was obtained by examining the correlation of B2 with the proton dependent processes of bacteriorhodopsin in buffers and dyes. Based on these observations, we suggest that most, if not all, of the intrinsic B2 component of the photocurrent at moderate salt concentration is due to proton release.

The photocurrents from purple membranes whose surface potential has been reduced by delipidation or chemical modification of carboxyl groups with methyl esters were found to be only modestly changed. This suggests that the salt effect is not through its modulation of the surface potential. Rather, we propose that in low salt B2 represents the sum of a proton release from the surface of the purple membrane and a second current component, due to cations moving back towards the membrane, which is only important in low salt. The cation counter current is induced by proton release which creates a transient uncompensated negative charge on the membrane.

     

Electrooptical studies on proton-binding and -release of bacteriorhodopsin

Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring the absorbance change of pH indicators. In connection with the photocycle and proton pump ability, three different states of bacteriorhodopsin were used: (1) the native purple bacteriorhodopsin (magnesium and calcium ions are bound, the M intermediate exists in the photocycle and protons are pumped), (2) the cation-depleted blue bacteriorhodopsin (no M intermediate), and (3) the regenerated purple bacteriorhodopsin which is produced either by raising the pH or by adding magnesium ions (the M intermediate exists). In the native purple bacteriorhodopsin there are, at least, two types of proton binding sites: one releases protons and the other takes up protons in the presence of the electric field. On the other hand, blue bacteriorhodopsin and the regenerated purple bacteriorhodopsin (pH increase) show neither proton release nor proton uptake. When magnesium ions are added to the suspensions; the field-induced pH change is observed again. Thus, the stability of proton binding depends strongly on the state of bacteriorhodopsin and differences in proton binding are likely to be related to differences in proton pump activity. Furthermore, it is suggested that the appearance of the M intermediate and proton pumping are not necessarily related.     

Effect of iodination of the purple membrane on the photocycle of bacteriorhodopsin

Bacteriorhodopsin in the purple membrane of Halobacterium halobium is coupled to a photocycle that results in the release and uptake of protons. The role of tyrosyl residues in the photocycle of bacteriorhodopsin has been investigated by the technique of chemical modifications of these residues by iodination and nitration. The studies indicate that modification of a tyrosyl residue accelerates M₄₁₂ formation, whereas modification of another type of tyrosine residue(s) accessible from the cytoplasmic surface of the purple membrane inhibits M₄₁₂ decay. The results support the hypothesis that a reversible deprotonation of tyrosine residues prior to and after M₄₁₂ formation in the photocycle are steps in the light-driven pathway of H⁺ translocation by bacteriorhodopsin.     

Surface Charge Movements of Purple Membrane During Light-Dark Adaptation

The difference in the surface charge distribution between light-adapted and dark-adapted purple membranes was investigated with electric dichroism measurements from approximately pH 5 to pH 11. Purple membrane sheets in solution are oriented in a weak electric field by their permanent dipole moment, which is due to the charge distribution of the membrane surfaces and/or within the membrane. The degree of orientation of purple membrane sheets was obtained from the measurement of “electrical anisotropy” of retinal chromophore in the membranes. At about pH 7, there was no difference in the “electric anisotropy” between light- and dark-adapted purple membranes. At about pH 9, the electric anisotropy of dark-adapted purple membrane was larger than that of light-adapted purple membrane. But at around pH 6 the difference was opposite. Linear dichroism experiments did not show any change of retinal tilt angle with respect to the membrane normal between the two forms from approximately pH 5 to pH 10. This result indicates that the changes in the “electric anisotropy” are not due to the change of retinal tilt angle, but due to the change in the permanent dipole moment of the membrane. To estimate the change in surface charges from the permanent dipole moment, we investigated the difference of the permanent dipole moment between the native purple membrane and papain-treated purple membrane in which negative charges in the cytoplasmic-terminal part are removed. This estimation suggests that this light-dark difference at around pH 9 can be accounted for by a change of ~0.5 electric charge per bacteriorhodopsin (bR) molecule at either of the two surfaces of the membrane. We also found from pH electrode measurements that at about pH 8 or 9 light adaptation was accompanied by an uptake of ~0.1 protons per bR. A possible movement of protons during light-dark adaptation is discussed. The direction of the permanent dipole moment does not change with papain treatment. The permanent dipole moment in papain-treated purple membrane is estimated to be 27 ±2 debye/bR.     

Flash-induced proton transfer in photosynthetic bacteria

A proton electrochemical potential across the membranes of photosynthetic purple bacteria is established by a light-driven proton pump mechanism: the absorbed light in the reaction center initiates electron transfer which is coupled to the vectorial displacement of protons from the cytoplasm to the periplasm. The stoichiometry and kinetics of proton binding and release can be tracked directly by electric (glass electrodes), spectrophotometric (pH indicator dyes) and conductimetric techniques. The primary step in the formation of the transmembrane chemiosmotic potential is the uptake of two protons by the doubly reduced secondary quinone in the reaction center and the subsequent exchange of hydroquinol for quinone from the membrane quinone-pool. However, the proton binding associated with singly reduced promary and/or secondary quinones of the reaction center is substoichiometric, pH-dependent and its rate is electrostatically enhanced but not diffusion limited. Molecular details of protonation are discussed based on the crystallographic structure of the reaction center of purple bacteriaRb. sphaeroides andRps. viridis, structure-based molecular (electrostatic) calculations and mutagenesis directed at protonatable amino acids supposed to be involved in proton conduction pathways.     

Newly isolated archaerhodopsin from a strain of Chinese halobacteria and its proton pumping behavior

A strain of extremely salt-loving halobacteria Halobacterium species xz515 from a salt lake in Tibet was isolated. SDS-polyacrylamide gel electrophoresis shows that there is only one protein on claret membrane, which is the same membrane fraction as purple membrane from Halobacterium salinarum, with a molecular weight close to bacteriorhodopsin (br). The purified retinal containing protein from xz515 has an absorption peak at around 550 nm. These facts indicate that it is a br-like protein. The partial sequence determination [H. Wang et al., Chin. Sci. Bull., 45 (2000)] shows that this br-like protein belongs to the archaerhodopsin family. The measurements of light-induced medium pH change in intact cells and cell envelope vesicles of xz515 suggest that this type of archaerhodopsin has a proton pumping function. However, the study about the dynamics of pumped protons across the membrane reveal that the proton release and proton uptake is in reverse order compared to br. The probable reason, attributing to regulating the rate of proton release is discussed.     

Influence of the surface potential on the purple membrane structure and activity

The role of the divalent cations in the purple membrane is generally understood as the release mechanism of the blue form appearance. The reconstitution by cation addition leads to the recovery of the initial spectral properties. Numerous data are available in the literature on this matter but they are scattered, so that synthetic understanding is not easy. The role of divalent cations was studied through spectrophotometric titrations and electrophoretic mobility measurements, i.e., zeta potential valuations. Thus, correlations between the bacteriorhodopsin (bR) state and the whole membrane in equilibrium with a definite medium could be made. Deionization was not a fully reversible process. The absence of cations affect neither the rate of the M412 formation nor its lifetime but the yield of M412/bR was 50% lower. The number of protons involved in the blue to purple transition of both membranes was different and the reconstitution did not erase this difference. It was observed that the number of protons dissociated upon cation addition corresponded approximately to the number of positive charges removed by deionization. Electrophoretic mobility titrations showed large differences between the membranes, illustrating the influence of the surface charge density on the pK of the transition. Taking advantage of the reversible light adaptation process, the reciprocal influence of the charge density of the membrane surface and the retinal state in bR was shown. Specificity of the divalent cations was questioned by a direct substitution of them by imidazol, which left the membrane intact. The partial reversibility of the deionization, the decrease of the M412 yield, the differences in the titratable protons, and the nonstrict specificity toward divalent cations suggested that another unknown factor could be removed from the membrane.     

Light-induced quinone reduction in photosystem II

The photosystem II core complex is the water:plastoquinone oxidoreductase of oxygenic photosynthesis situated in the thylakoid membrane of cyanobacteria, algae and plants. It catalyzes the light-induced transfer of electrons from water to plastoquinone accompanied by the net transport of protons from the cytoplasm (stroma) to the lumen, the production of molecular oxygen and the release of plastoquinol into the membrane phase. In this review, we outline our present knowledge about the "acceptor side" of the photosystem II core complex covering the reaction center with focus on the primary (Q(A)) and secondary (Q(B)) quinones situated around the non-heme iron with bound (bi)carbonate and a comparison with the reaction center of purple bacteria. Related topics addressed are quinone diffusion channels for plastoquinone/plastoquinol exchange, the newly discovered third quinone Q(C), the relevance of lipids, the interactions of quinones with the still enigmatic cytochrome b559 and the role of Q(A) in photoinhibition and photoprotection mechanisms. This article is part of a Special Issue entitled: Photosystem II.     

Characterization of metal ion-binding sites in bacteriorhodopsin

We have investigated the effects of the binding of various metal ions to cation-free bacteriorhodopsin ("blue membrane"). The following have been measured: shift of the absorption maximum from 603 to 558 nm (blue to purple transition), binding isotherms, the release of H+ upon binding, and the decay of the deprotonated intermediate of the photocycle, M412. We find that all cations of the lanthanide series, as well as the alkali and alkali earth metals earlier investigated, are able to bring about the absorption shift, whereas Hg2+ and Pt4+ are not. Sigmoidal spectroscopic titration curves and nonsigmoidal binding curves suggest that there are two high affinity sites for cations in bacteriorhodopsin. Binding to the site with the second highest affinity is responsible for the absorption shift. Divalent cation binding to blue membrane causes release of about six protons, whereas higher numbers of protons are released by trivalent cations, suggesting that the shift of absorption maximum involves proton release from carboxyl group(s). The metal ion bound to this site must be surrounded by carboxyl oxygen atoms acting together as a multidentate ligand with a specific geometry because multivalent ions are effective only when capable of octahedral coordination. Lanthanide ions dramatically inhibit M412 decay at pH above 6.3, an effect probably due to binding to lipid phosphoryl groups.     

Relationship of proton release at the extracellular surface to deprotonation of the schiff base in the bacteriorhodopsin photocycle.

The surface potential of purple membranes and the release of protons during the bacteriorhodopsin photocycle have been studied with the covalently linked pH indicator dye, fluorescein. The titration of acidic lipids appears to cause the surface potential to be pH-dependent and causes other deviations from ideal behavior. If these anomalies are neglected, the appearance of protons can be followed by measuring the absorption change of fluorescein bound to various residues at the extracellular surface. Contrary to widely held assumption, the activation enthalpies of kinetic components, deuterium isotope effects in the time constants, and the consequences of the D85E, F208R, and D212N mutations demonstrate a lack of direct correlation between proton transfer from the buried retinal Schiff base to D85 and proton release at the surface. Depending on conditions and residue replacements, the proton release can occur at any time between the protonation of D85 and the recovery of the initial state. We conclude that once D85 is protonated the proton release at the extracellular protein surface is essentially independent of the chromophore reactions that follow. This finding is consistent with the recently suggested version of the alternating access mechanism of bacteriorhodopsin, in which the change of the accessibility of the Schiff base is to and away from D85 rather than to and away from the extracellular membrane surface.     

Large transient nonproton ion movements in purple membrane suspensions are abolished by solubilization in Triton X-100.

Light-induced release/uptake of both protons and other ions cause transient changes in conductivity in suspensions of purple membrane (PM) fragments (Marinetti, Tim, and David Mauzerall, 1983, Proc. Natl. Acad. Sci. USA, 80:178-180). We find that the release/uptake of nonproton ions with quantum yield greater than 1 is observed at most pHs and ionic strengths. Only at both low pH and low ionic strength is the conductivity transient mostly due to protons. Our hypothesis is that during the photocycle, changes occur in the PM's dense surface charge distribution that result in changes in the number of counterions bound or condensed at the membrane surface. To test this, the PM structure was perturbed with the nonionic detergent Triton X-100. Immediately after addition, Triton does not abolish the nonproton ion movements; in fact at low detergent concentrations (0.02% vol/vol) the signal amplitudes increased considerably. However, when PM is completely solubilized into monomers in Triton, the conductivity transients are due to protons alone, though at lower quantum yield compared with native PM. These results suggest that changes in the surface charge distribution in native PM's photocycle could contribute to proton transfer between the aqueous phase and bR itself.     

Light-driven proton translocations in Halobacterium halobium.

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating electrochemical proton gradient across the cell membrane. However, the type response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium. The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow. The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N'-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor. Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.     

Large scale nonproton ion release and bacteriorhodopsin's state of aggregation in lipid vesicles. I. Monomers.

Light-induced conductivity transients have been observed in preparations of bacteriorhodopsin (bR) in phospholipid vesicles at high lipid/protein molar ratios. Under these conditions, bR is known to be dissolved as monomers in the lipid bilayer. The conductivity transients are due mostly to proton movements, including a trans-membrane component. Kinetic resolution of the conductance change due to proton ionophore-induced leakage through the vesicle membrane provides a novel method to quantitate the number of protons pumped, even in heavily buffered solutions. Some of the transient signal seen on the timescale of the bR photocycle is due to nonproton ions but is smaller than that observed in native purple membranes at pH 7 in low salt. Furthermore, when the pH is raised to 8, the very large transient nonproton ion release seen in purple membranes is not seen in the vesicles. This correlates well with previous results (Marinetti, T., and D. Mauzerall, 1986, Biophys. J., 50:405-415), in which the nonproton ion movements observed with native purple membranes were abolished by solubilization in Triton X-100. Thus, the nonproton ion release appears to be a property of bR in the native aggregated state.     

Water molecules and exchangeable hydrogen ions at the active centre of bacteriorhodopsin localized by neutron diffraction. Elements of the proton pathway?

Neutron diffraction is used to localize water molecules and/or exchangeable hydrogen ions in the purple membrane by H2O/2H2O exchange experiments at different values of relative humidity. At 100% relative humidity, differences in the hydration between protein and lipid areas are observed, accounting for an excess amount of about 100 molecules of water in the lipid domains per unit cell. A pronounced isotope effect was observed, reproducibly showing an increase in the lamellar spacing from 60 A in 2H2O to 68 A in H2O. At 15% relative humidity, the positions of exchangeable protons became visible. A dominant difference density peak corresponding to 11 +/- 2 exchangeable protons was detected in the central part of the projected structure of bacteriorhodopsin at the Schiff's base end of the chromophore. A difference density map obtained from data on purple membrane films at 15% relative humidity in 2H2O, and the same sample after complete drying in vacuum, revealed that about eight of these protons belong to four water molecules. This is direct evidence for tightly bound water molecules close to the chromophore binding site of bacteriorhodopsin, which could participate in the active steps of H+ translocation as well as in the proton pathway across this membrane protein.     

Subsecond proton-hole propagation in bacteriorhodopsin

The dynamics of proton transfer between the surface of purple membrane and the aqueous bulk have recently been investigated by the Laser Induced Proton Pulse Method. Following a Delta-function release of protons to the bulk, the system was seen to regain its state of equilibrium within a few hundreds of microseconds. These measurements set the time frame for the relaxation of any state of acid-base disequilibrium between the bacteriorhodopsin's surface and the bulk. It was also deduced that the released protons react with the various proton binding within less than 10 micro s. In the present study, we monitored the photocycle and the proton-cycle of photo-excited bacteriorhodopsin, in the absence of added buffer, and calculated the proton balance between the Schiff base and the bulk phase in a time-resolved mode. It was noticed that the late phase of the M decay (beyond 1 ms) is characterized by a slow (subsecond) relaxation of disequilibrium, where the Schiff base is already reprotonated but the pyranine still retains protons. Thus, it appears that the protonation of D96 is a slow rate-limiting process that generates a "proton hole" in the cytoplasmic section of the protein. The velocity of the hole propagation is modulated by the ionic strength of the solution and by selective replacements of charged residues on the interhelical loops of the protein, at domains that seems to be remote from the intraprotein proton conduction trajectory.     

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.