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.     

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.     

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.     

Actinic light density dependence of the bacteriorhodopsin protocycle.

The photocycle of bacteriorhodopsin (BR) was studied in the 0.3 microsecond to 10 s time interval after excitation, using a wide range of actinic light intensities (10 ns half-duration, 0.06-60 mJ/cm2), at neutral and alkaline pH values. The relative weights of the rapidly and the slowly decaying components of the M intermediate (Mf and M(s), respectively) and the yield of the third millisecond component, N(R,P), are the function of the exciting light intensity (density), while their lifetimes are not. The relative weight of M(s) is found to be a linear function of the portion of the BR molecules undergoing the photocycle. This suggests the existence of a cooperative interaction of the BR molecules arranged in the crystalline purple membrane sheets. Another source of M(s) is also found, which results a nonvanishing relative weight of M(s) even at very weak actinic light density values. The explanation for this may be a branching, or the heterogeneity of BR itself or with its environment. It is shown that the relative weights of the rising and decaying components of the M form(s) do not correlate directly with each other.     

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.     

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.     

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。     

pH dependence of light-driven proton pumping by an archaerhodopsin from Tibet: comparison with bacteriorhodopsin

The pH-dependence of photocycle of archaerhodopsin 4 (AR4) was examined, and the underlying proton pumping mechanism investigated. AR4 is a retinal-containing membrane protein isolated from a strain of halobacteria from a Tibetan salt lake. It acts as a light-driven proton pump like bacteriorhodopsin (BR). However, AR4 exhibits an "abnormal" feature--the time sequence of proton release and uptake is reversed at neutral pH. We show here that the temporal sequence of AR4 reversed to "normal"--proton release preceding proton uptake--when the pH is increased above 8.6. We estimated the pK(a) of the proton release complex (PRC) in the M-intermediate to be approximately 8.4, much higher than 5.7 of wide-type BR. The pH-dependence of the rate constant of M-formation shows that the pK(a) of PRC in the initial state of AR4 is approximately 10.4, whereas it is 9.7 in BR. Thus in AR4, the chromophore photoisomerization and subsequent proton transport from the Schiff base to Asp-85 is coupled to a decrease in the pK(a) of PRC from 10.4 to 8.4, which is 2 pK units less than in BR (4 units). This weakened coupling accounts for the lack of early proton release at neutral pH and the reversed time sequence of proton release and uptake in AR4. Nevertheless the PRC in AR4 effectively facilitates deprotonation of primary proton acceptor and recovery of initial state at neutral pH. We found also that all pK(a)s of the key amino acid residues in AR4 were elevated compared to those of BR.     

Deprotonation of the Schiff base of bacteriorhodopsin is obligate in light-induced proton pumping

Bacteriorhodopsin (bR) in purple membranes was permethylated with formaldehyde and pyridine-borane with the incorporation of approximately 12 methyl groups. This new pigment, PMbR, absorbed light in the dark-adapted state with a lambda max at 558 nm, virtually the same as that of bR. Light adaptation of PMbR produced a lambda max of 564 nm with a slightly elevated epsilon. Similar changes occurred with bR. When incorporated into asolectin vesicles, PMbR was able to pump protons in the light with an efficiency similar to that of bR itself. Bleaching of PMbR exposed its active site lysine residue, which was monomethylated to form active site methylated bR (AMbR) after regeneration with all-trans-retinal. This blue pigment, which is a cyanopsin rather than a rhodopsin, showed an extraordinary red shift, absorbing light with a lambda max of 620 nm in the dark-adapted state. Light adaptation of AMbR resulted in a spectral shift to 616 nm with a decrease in epsilon. This change was completely reversible in the dark. This shift was interpreted to mean that an L-like intermediate was accumulating, as would be expected if deprotonation of the protonated Schiff base could not occur to produce the M intermediate. Furthermore, when incorporated into asolectin vesicles, AMbR proved incapable of pumping protons in the light. It was concluded from these experiments that deprotonation of the Schiff base of bR is obligate for light-induced proton pumping.     

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.     

Thr90 is a key residue of the bacteriorhodopsin proton pumping mechanism.

Mutation of Thr90 to Ala has a profound effect on bacteriorhodopsin properties. T90A shows about 20% of the proton pumping efficiency of wild type, once reconstituted into liposomes. Mutation of Thr90 influences greatly the Schiff base/Asp85 environment, as demonstrated by altered lambda(max) of 555 nm and pK(a) of Asp85 (about 1.3 pH units higher than wild type). Hydroxylamine accessibility is increased in both dark and light and differential scanning calorimetry and visible spectrophotometry show decreased thermal stability. These results suggest that Thr90 has an important structural role in both the unphotolysed bacteriorhodopsin and in the proton pumping mechanism.     

Effect of xenon binding to a hydrophobic cavity on the proton pumping cycle in bacteriorhodopsin

To understand the functional role of apolar cavities in bacteriorhodopsin, a light-driven proton pump found in Halobacterium salinarum, we investigated the crystal structure in pressurized xenon or krypton. Diffraction data from the P622 crystal showed that one Xe or Kr atom binds to a preexisting hydrophobic cavity buried between helices C and D, located at the same depth from the membrane surface as Asp96, a key residue in the proton uptake pathway. The occupation fraction of Xe or Kr was calculated as approximately 0.32 at a pressure of 1 MPa. In the unphotolyzed state, the binding of Xe or Kr caused no large deformation of the cavity. However, the proton pumping cycle was greatly perturbed when an aqueous suspension of purple membrane was pressurized with xenon gas; that is, the decay of the M state was accelerated significantly (~5 times at full occupancy), while the decay of an equilibrium state of N and O was slightly decelerated. A similar but much smaller perturbation in the reaction kinetics was observed upon pressurization with krypton gas. In a glycerol/water mixture, xenon-induced acceleration of M decay became less significant in proportion to the water activity. Together with the structure of the xenon-bound protein, these observations suggest that xenon binding helps water molecules permeate into apolar cavities in the proton uptake pathway, thereby accelerating the water-mediated proton transfer from Asp96 to the Schiff base.     

Inhibitors of proton pumping: effect on passive proton transport

Reported inhibitors of the Characean plasmalemma proton pump were tested for their ability to inhibit the passive H⁺ conductance which develops in Chara corallina Klein ex Willd. at high pH. Diethylstilbestrol inhibits the proton pump and the passive H⁺ conductance with about the same time course, at concentrations that have no effect on cytoplasmic streaming. N-Ethylmaleimide, a sulfhydryl reagent which is small and relatively nonpolar, also inhibits both pumping and passive conductance of H⁺. However, it also inhibits cytoplasmic streaming with about the same time course, and therefore could not be considered a specific ATPase inhibitor. p-Chloromercuribenzene sulfonate (PCMBS), a sulfhydryl reagent which is large and charged and hence less able to penetrate the membrane, does not inhibit pumping or conductance at low concentration. At high concentration, PCMBS sometimes inhibits pumping without affecting H⁺ conductance, but since streaming is also inhibited, the effect on the pump cannot be said to be specific. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, a water soluble carbodiimide, weakly inhibits both pump and conductance, apparently specifically.     

Regulation of the bacteriorhodopsin photocycle and proton pumping in whole cells of Halobacterium salinarium.

Single-turnover kinetics of the bacteriorhodopsin photocycle and proton-pumping capabilities of whole cells were studied. It was found that the Delta mu (tilde)H+ of the cell had a profound influence on the kinetics and components of the cycle. For example, comparing the photocycle in whole cells to that seen in PM preparations, we found that (1) the single-turnover time of the cycle was increased approximately 10-fold, (2) the mole fraction of M-fast (at high actinic light) decreased from 50 to 20%, and (3) the time constant for M-slow increased significantly. The level of Delta mu(tilde)H+ was dependent on respiration, ATP formation and breakdown, and the magnitude of a pre-existing K+ diffusion gradient. The size of the Delta mu(tilde)H+ could be manipulated by additions of HCN, nigericin, and DCCD (N,N'-dicyclohexylcarbodamide). At higher levels of Delta mu(tilde)H+, further changes in the photocycle were seen. (4) Two slower components of M-decay appeared as major components. (5) The apparent conversion of the M-fast to the O intermediate disappeared. (6) A partial reversal of an early photocycle step occurred. The photocycle of intact cells could be changed to that seen in purple membrane suspensions by the energy-uncoupler CCCP or by lysis of the cells. In fresh whole cells, light-induced proton pumping was not seen until the K+ diffusion potential was dissipated and proton accumulation facilitated by use of a K+-H+ exchanger (nigericin), respiration was inhibited by HCN, and ATP synthesis and breakdown were inhibited by DCCD. In stored cells, the pre-existing K+ diffusion gradient was diminished through slow diffusion, and only DCCD and HCN were required to elicit proton extrusion.     

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.     

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 alpha-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相似文献   

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.     

Trans/13-cis isomerization is essential for both the photocycle and proton pumping of bacteriorhodopsin.

We studied an analogue of bacteriorhodopsin whose chromophore is based on all-trans retinal. A five-membered ring was built around the 13-14 double bond so as to prohibit trans to 13-cis isomerization. No light-induced photochemical changes were seen, other than those due to a small amount (approximately 5%) of unbleached bacteriorhodopsin remaining in the apomembrane used for regeneration. The techniques used included flash photolysis at room and liquid nitrogen temperatures and Fourier-transform infrared difference spectroscopy. When the trans-fixed pigment was incorporated into phospholipid vesicles, no evidence of light-initiated proton pumping could be found. The results indicate that trans to 13-cis isomerization is essential for the photochemical transformation and function of bacteriorhodopsin.