Events in proton pumping by bacteriorhodopsin.

The short-circuit photoresponse of a bacteriorhodopsin-based photoactive membrane is studied. The membrane is formed by first coating a Teflon membrane with lipid and then fusing bacteriorhodopsin vesicles to it. An incandescent light source was used to obtain the rise time of the photocurrent in response to a step-function illumination. A fast response, less than 1 ms, characterizes the initial rise and decay of the photocurrent. The trailing edge of the rise and trailing edge of the decay each exhibit different time constants both greater than 1 ms. These slower components show a sensitivity to membrane charging, the presence of diethylether in the bathing solution, and the presence of a charged cation complex in the lipid region. The photoresponse is not analyzed by means of the usual equivalent electrical circuit, but rather in terms of image charges in the conducting electrolyte bathing the membrane. Further experiments using a pulsed laser (pulse width less than 1 microseconds) resolve at least three time constants in the photoresponse: 0.057 ms, 1.06 ms, and 13 ms. Three distinct charge displacements (4.4, 7.5, and 33.1 A) are derived from the data, each corresponding to one of the above time constants.     

Actinic light and pH effect on the proton pumping of bacteriorhodopsin

The actinic light effect on the bacteriorhodopsin (BR) photocycle kinetics led to the assumption of a cooperative interaction between the photocycling BR molecules. In this paper we report the results of the actinic light effect and pH on the proton release and uptake kinetics. An electrical method is applied to detect proton release and uptake during the photocycle [E. Papp, G. Fricsovszky, J. Photochem. Photobiol. B: Biol. 5 (1990) 321]. The BR photocycle kinetics was also studied by absorption kinetics measurements at 410 nm and the data were analyzed by the local analysis of the M state kinetics [E. Papp, V.H. Ha, Biophys. Chem. 57 (1996) 155]. While at high pH and ionic strength, we found a similar behavior as reported earlier, at low ionic strength the light effect proved to be more complex. The main conclusions are the following: Though the number of BR excited to the photocycle (fraction cycling, fc) goes to saturation with increasing laser pulse energy, the absorbed energy by BR increases linearly with pulse energy. From the local analysis we conclude that the light effect changes the kinetics much earlier, already at the L intermediate state decay. The transient electric signal, caused by proton release and uptake, can be decomposed into two components similarly to the absorption kinetic data of the M intermediate state. The actinic light energy affects mainly the ratio of the two components and the proton movements inside BR while pH has an effect on the kinetics of the proton release and uptake groups at the membrane surface.     

细菌视紫红质中质子泵出的分子机理

细菌视紫红质(Bacteriorhodopsin,或bR)是盐生嗜盐菌(Halobacterium salinarium)等细菌的跨膜蛋白质,其色基视黄醛的光致异构化作用触发细菌视紫红质的一系列结构变化,把质子从细胞质泵到细胞外空间。对细菌视紫红质中质子泵出分子机理进行了描述。     

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.     

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.     

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.     

Quantum efficiency of light-driven proton extrusion in Halobacterium halobium. pH dependence.

The quantum yield for light-induced proton extrusion in Halobacterium halobium cells pretreated with an ATPase inhibitor was measured between pH 5 and 9 using two separate spectrophotometric techniques. The transmittance of the cell suspension was measured with a spectrometer equipped with "end-on" photomultipliers, whereas the reflectance was measured using a light-integrating sphere. The potentialities of the two techniques are critically compared. These measurements are used to evaluate the intensities of light absorbed by the cells. Since the initial rates of proton release into the extracellular medium were simultaneously measured, the quantum yield values [QY(H+)] could be determined. The results obtained with the two techniques are in reasonable agreement. QY(H+) is 0.64 at pH 5.9 and decreases gradually to 0.28 at alkaline pH values.     

The effect of pH on proton transport by bacteriorhodopsin

The pH-dependence of proton motion during the photocycle was investigated by measuring the photoelectric signals due to charge displacement inside bacteriorhodopsin molecules. Measurements were performed on purple membranes oriented in suspension and the kinetics of flash excited electric and light absorption signals was compared. It was found that in the pH range 4.5–8 the photocycle and the successive proton movements have identical kinetics, and do not depend on pH. In the pH range 8–10 both kinetics change, though differently; the charge motion decouples from the photocycle and the photocycle seems to split up into two parallel paths, the photoelectric signal becomes faster. However, the net proton transfer remains the same as at lower pH values. Above pH ≈ 10, the photocycle behaves differently and cannot be described by the parallel pathway model and the net proton displacement drops. The results are explained by the successive titration of two groups (probably tyrosine) participating in proton translocation.     

Triton X-100 can alter the temporal sequence of the light-driven proton pump of archaerhodopsin 4

We report that Triton X-100 can alter the temporal sequence of the light-induced proton uptake and release of archaerhodopsin 4 (AR4), a proton pumping protein in a species of Halobacteria from a Tibetan salt lake. Under physiological conditions, AR4 isolated from the bacterium exhibits a reversed temporal order of proton release and uptake compared to what is observed for bacteriorhodopsin (BR). However, in the presence of Triton X-100 early proton release was observed in AR4 at neutral pH by us. Further, this temporal order for light-driven proton release and uptake for AR4 was found to be recovered after the removal of Triton X-100 by Biobeads. This phenomenon of detergent-induced alteration of the order of proton release and uptake has not yet been reported in any other retinal-containing membrane protein such as BR. Our findings indicate that the function of AR4 is influenced by its self-assembled state, and meanwhile imply some subtle protein-lipid interactions or protein-protein interactions in adjusting the proton pumping behavior of AR4.     

pH dependence of bacteriorhodopsin thermal unfolding

The thermal denaturation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by differential scanning calorimetry (DSC) and temperature-dependent spectroscopy in the pH range from 5 to 11. Monitoring of protein fluorescence and absorbance in the near-UV and visible regions indicates that changes primarily occur in tertiary structure with denaturation. Far-UV circular dichroism shows only small changes in the secondary structure, unlike most globular water-soluble proteins of comparable molecular weight. The DSC transition can best be described as a two-state denaturation of the trimer. Thermodynamic analysis of the calorimetric transition reveals some similarity between the unfolding of bacteriorhodopsin and water-soluble proteins. Specifically, a pH dependence of the midpoint temperature of denaturation is seen as well as a temperature-dependent enthalpy of denaturation. Proteolysis experiments on denatured purple membrane suggest that bacteriorhodopsin may be partially extruded from the membrane as it denatures. Exposure of buried hydrophobic residues to the aqueous environment upon denaturation is consistent with the observed temperature-dependent enthalpy.     

The voltage-dependent proton pumping in bacteriorhodopsin is characterized by optoelectric behavior

The light-driven proton pump bacteriorhodopsin (bR) was functionally expressed in Xenopus laevis oocytes and in HEK-293 cells. The latter expression system allowed high time resolution of light-induced current signals. A detailed voltage clamp and patch clamp study was performed to investigate the DeltapH versus Deltapsi dependence of the pump current. The following results were obtained. The current voltage behavior of bR is linear in the measurable range between -160 mV and +60 mV. The pH dependence is less than expected from thermodynamic principles, i.e., one DeltapH unit produces a shift of the apparent reversal potential of 34 mV (and not 58 mV). The M(2)-BR decay shows a significant voltage dependence with time constants changing from 20 ms at +60 mV to 80 ms at -160 mV. The linear I-V curve can be reconstructed by this behavior. However, the slope of the decay rate shows a weaker voltage dependence than the stationary photocurrent, indicating that an additional process must be involved in the voltage dependence of the pump. A slowly decaying M intermediate (decay time > 100 ms) could already be detected at zero voltage by electrical and spectroscopic means. In effect, bR shows optoelectric behavior. The long-lived M can be transferred into the active photocycle by depolarizing voltage pulses. This is experimentally demonstrated by a distinct charge displacement. From the results we conclude that the transport cycle of bR branches via a long-lived M(1)* in a voltage-dependent manner into a nontransporting cycle, where the proton release and uptake occur on the extracellular side.     

Voltage dependence of proton pumping by bacteriorhodopsin is regulated by the voltage-sensitive ratio of M1 to M2.

The voltage dependence of light-induced proton pumping was studied with bacteriorhodopsin (bR) from Halobacterium salinarum, expressed in the plasma membrane of oocytes from Xenopus laevis in the range -160 mV to +60 mV at different light intensities. Depending on the applied field, the quenching effect by blue light, which bypasses the normal photo and transport cycle, is drastically increased at inhibiting (negative) potentials, and is diminished at pump current increasing (positive) potentials. At any potential, two processes with different time constants for the M --> bR decay of approximately 5 ms (tau1) and approximately 20 ms (tau2) are obtained. At pump-inhibiting potentials, a third, long-lasting process with tau3 approximately 300 ms at neutral pH is observed. The fast processes (tau1, tau2) can be assigned to the decay of M2 in the normal pump cycle, i.e., to the reprotonation of the Schiff base via the cytoplasmic side, whereas tau3 is due to the decay of M1 without net pumping, i.e., the reprotonation of the Schiff base via the extracellular side. The results are supported by determination of photocurrents induced by bR on planar lipid films. The pH dependence of the slow decay of M1 is fully in agreement with the interpretation that the reprotonation of the Schiff base occurs from the extracellular side. The results give strong evidence that an externally applied electrical field changes the ratio of the M1 and the M2 intermediate. As a consequence, the transport cycle branches into a nontransporting cycle at negative potentials. This interpretation explains the current-voltage behavior of bR on a new basis, but agrees with the isomerisation, switch, transfer model for vectorial transport.     

External electric control of the proton pumping in bacteriorhodopsin

Comparative analysis of the photoelectric response of dried films of purple membranes (PM) depending on their degree of orientation is presented. Time dependence of the photo-induced protein electric response signal (PERS) of oriented and non-oriented films to a single laser pulse in the presence of the external electric field (EEF) was experimentally determined. The signal does not appear in the non-oriented films when the EEF is absent, whereas the PERS of the oriented PM films demonstrates the variable polarity on the microsecond time scale. In the presence of the EEF the PERS of the non-oriented film rises exponentially preserving the same polarization. The polarization of the PERS changes by changing the polarity of the EEF with no influence on the time constant of the PERS kinetics. The EEF effect on the PERS of the oriented films is more complicated. By subtracting the PERS when EEF ≠ 0 from the PERS when EEF = 0 the resulting signal is comparable to that of the non-oriented films. Generalizing the experimental data we conclude that the EEF influence is of the same origin for the films of any orientation. To explain the experimental results the two-state model is suggested. It assumes that the EEF directionally changes the pK_a values of the Schiff base (SB) and of the proton acceptor aspartic acid D85 in bacteriorhodopsin. Because of that the SB→D85 proton transfer might be blocked and consequently the L→M intermediate transition should vanish. Thus, on the characteristic time scale τ _{L → M} ≈ 30 μs; both intermediates, the M intermediate, appearing under normal conditions, and the L intermediate as persisting under the blocked conditions when D85 is protonated, should coexist in the film. The total PERS is a result of the potentials corresponding to the electrogenic products of intermediates L and M that are of the opposite polarity. It is concluded that the ratio of bacteriorhodopsin concentrations corresponding to the L and M intermediates is driven by the EEF and, consequently, it should define the PERS of the non-oriented films. According to this model the orientation degree of the film could be evaluated by describing the PERS.     

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.     

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.     

The possible role of the closed-open transition in proton pumping by cytochrome c oxidase: the pH dependence of cyanide inhibition

The rate of oxidation of reduced cytochrome c catalyzed by cytochrome oxidase in the presence and absence of cyanide has been measured spectrophotometrically at pH 5.5, 6.4, 7.4 and 8.3. At the cytochrome c concentration used (272 microM), the uninhibited rate is maximal at pH 6.4 and drops to a value about one sixth of this maximum at pH 8.3. In the presence of cyanide, the rate initially drops rapidly, but with the cyanide concentration used (5.5 microM) there is still a measurable rate of oxidation when maximal inhibition has been reached. This inhibited rate decreases as the pH increases, whereas the apparent rate constant for cyanide binding is almost independent of pH. The results have been analyzed on the basis of a model in which two-electron reduction of the oxidized enzyme triggers a transition from a closed to an open conformation. It is assumed that cyanide can only bind to the open conformation and, furthermore, that rapid internal electron transfer to the dioxygen-reducing site occurs in this state alone. The analysis shows that the true constant for cyanide binding decreases with decreasing pH to a constant value at low pH. It also indicates that the increase in the catalytic constant with decreasing pH is associated with an increase in the rate of the closed-open conformational transition on protonation of the enzyme, and it is proposed that this transition is operative in electron gating in the proton-pump function of the enzyme.     

pH dependence of proton translocation by Escherichia coli.

Proton translocation in spheroplasts from Escherichia coli has been studied in two mutants, one of which expresses cytochrome o and the other cytochrome d as the terminal oxidase. Using the O2 pulse method, the H+/e- ratio of proton translocation associated with cytochrome o was confirmed to be near 2 at neutral pH, but was found to decrease considerably when the medium pH was raised above 8. At high pH there was an increase in H+/OH- permeability of the cell membrane, but this was not sufficient to explain the decline in proton ejection. The pH effect was confined to cytochrome o-linked activity. It was not present when cytochrome d generated the electrochemical proton gradient. This makes it improbable that the Na+/H+ antiporter is responsible. The most likely explanation for our finding is that there is a "slip" in the proton-pumping mechanism of cytochrome o at high pH.     

Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning α-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of “the cation” in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca²⁺, Mg²⁺ and Mn²⁺) and monovalent metal cations (Li⁺, Na⁺, K⁺ and Cs⁺), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation<monovalent amine anesthetic cation<divalent metal cation. We found that organic cations such as the amine anesthetics can also regenerate the proton pump in the bR protein. The inhibition of proton transport in the bR protein by the anesthetic cations was elucidated using the wild type, the E204Q and the D96N mutated bRs. The hydrophobic interaction of the amine anesthetics with the bR protein plays an important part in inhibiting the bR proton pump.