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1.
The factors determining the pH dependence of the formation and decay of the O photointermediate of the bacteriorhodopsin (bR) photocycle were investigated in the wild-type (WT) pigment and in the mutants of Glu-194 and Glu-204, key residues of the proton release group (PRG) in bR. We have found that in the WT the rate constant of O --> bR transition decreases 30-fold upon decreasing the pH from 6 to 3 with a pKa of about 4.3. D2O slows the rise and decay of the O intermediate in the WT at pH 3.5 by a factor of 5.5. We suggest that the rate of the O --> bR transition (which reflects the rate of deprotonation of the primary proton acceptor Asp-85) at low pH is controlled by the deprotonation of the PRG. To test this hypothesis, we studied the E194D mutant. We show that the pKa of the PRG in the ground state of the E194D mutant, when Asp-85 is protonated, is increased by 1.2 pK units compared to that of the WT. We found a similar increase in the pKa of the rate constant of the O --> bR transition in E194D. This provides further evidence that the rate of the O --> bR transition is controlled by the PRG. In a further test, the E194Q mutation, which disables the PRG and slows proton release, almost completely eliminates the pH dependence of O decay at pHs below 6. A second phenomenon we investigated was that in the WT at neutral and alkaline pH the fraction of the O intermediate decreases with pKa 7.5. A similar pH dependence is observed in the mutants in which the PRG is disabled, E194Q and E204Q, suggesting that the decrease in the fraction of the O intermediate with pKa ca. 7.5 is not controlled by the PRG. We propose that the group with pKa 7.5 is Asp-96. The slowing of the reprotonation of Asp-96 at high pH is the cause of the decrease in the rate of the N --> O transition, leading to the decrease in the fraction of O.  相似文献   

2.
Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 &mgr;s to 75 &mgr;s with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.  相似文献   

3.
Tyrosine-83, a residue which is conserved in all halobacterial retinal proteins, is located at the extracellular side in helix C of bacteriorhodopsin. Structural studies indicate that its hydroxyl group is hydrogen bonded to Trp189 and possibly to Glu194, a residue which is part of the proton release complex (PRC) in bacteriorhodopsin. To elucidate the role of Tyr83 in proton transport, we studied the Y83F and Y83N mutants. The Y83F mutation causes an 11 nm blue shift of the absorption spectrum and decreases the size of the absorption changes seen upon dark adaptation. The light-induced fast proton release, which accompanies formation of the M intermediate, is observed only at pH above 7 in Y83F. The pK(a) of the PRC in M is elevated in Y83F to about 7.3 (compared to 5.8 in WT). The rate of the recovery of the initial state (the rate of the O --> BR transition) and light-induced proton release at pH below 7 is very slow in Y83F (ca. 30 ms at pH 6). The amount of the O intermediate is decreased in Y83F despite the longer lifetime of O. The Y83N mutant shows a similar phenotype in respect to proton release. As in Y83F, the recovery of the initial state is slowed several fold in Y83N. The O intermediate is not seen in this mutant. The data indicate that the PRC is functional in Y83F and Y83N but its pK(a) in M is increased by about 1.5 pK units compared to the WT. This suggests that Tyr83 is not the main source for the proton released upon M formation in the WT; however, Tyr83 is involved in the proton release affecting the pK(a) of the PRC in M and the rate of proton transport from Asp85 to PRC during the O --> bR transition. Both the Y83F and the Y83N mutations lead to a greatly decreased functionality of the pigment at high pH because most of the pigment is converted into the inactive P480 species, with a pK(a) 8-9.  相似文献   

4.
The effects of pH on the yield (phi(r)), and on the apparent rise and decay constants (k(r), k(d)), of the O(630) intermediate are important features of the bacteriorhodopsin (bR) photocycle. The effects are associated with three titration-like transitions: 1) A drop in k(r), k(d), and phi(r) at high pH [pK(a)(1) approximately 8]; 2) A rise in phi(r) at low pH [pK(a)(2) approximately 4.5]; and 3) A drop in k(r) and k(d) at low pH [pK(a)(3) approximately 4. 5]. (pK(a) values are for native bR in 100 mM NaCl). Clarification of these effects is approached by studying the pH dependence of phi(r), k(r), and k(d) in native and acetylated bR, and in its D96N and R82Q mutants. The D96N experiments were carried out in the presence of small amounts of the weak acids, azide, nitrite, and thiocyanate. Analysis of the mutant's data leads to the identification of the protein residue (R(1)) whose state of protonation controls the magnitude of phi(r), k(r), and k(d) at high pH, as Asp-96. Acetylation of bR modifies the Lys-129 residue, which is known to affect the pK(a) of the group (XH), which releases the proton to the membrane exterior during the photocycle. The effects of acetylation on the O(630) parameters reveal that the low-pH titrations should be ascribed to two additional protein residues R(2) and R(3). R(2) affects the rise of phi(r) at low pH, whereas the state of protonation of R(3) affects both k(r) and k(d). Our data confirm a previous suggestion that R(3) should be identified as the proton release moiety (XH). A clear identification of R(2), including its possible identity with R(3), remains open.  相似文献   

5.
In wild-type bacteriorhodopsin light-induced proton release occurs before uptake at neutral pH. In contrast, in mutants in which R82 is replaced by a neutral residue (as in R82A and R82Q), only a small fraction of the protons is released before proton uptake at neutral pH; the major fraction is released after uptake. In R82Q the relative amounts of the two types of proton release, "early" (preceding proton uptake) and "late" (following proton uptake), are pH dependent. The main conclusions are that 1) R82 is not the normal light-driven proton release group; early proton release can be observed in the R82Q mutant at higher pH values, suggesting that the proton release group has not been eliminated. 2) R82 affects the pKa of the proton release group both in the unphotolyzed state of the pigment and during the photocycle. In the wild type (in 150 mM salt) the pKa of this group decreases from approximately 9.5 in the unphotolyzed pigment to approximately 5.8 in the M intermediate, leading to early proton release at neutral pH. In the R82 mutants the respective values of pKa of the proton release group in the unphotolyzed pigment and in M are approximately 8 and 7.5 in R82Q (in 1 M salt) and approximately 8 and 6.5 in R82K (in 150 mM KCl). Thus in R82Q the pKa of the proton release group does not decrease enough in the photocycle to allow early proton release from this group at neutral pH. 3) Early proton release in R82Q can be detected as a photocurrent signal that is kinetically distinct from those photocurrents that are due to proton movements from the Schiff base to D85 during M formation and from D96 to the Schiff base during the M-->N transition. 4) In R82Q, at neutral pH, proton uptake from the medium occurs during the formation of O. The proton is released during the O-->bacteriorhodopsin transition, probably from D85 because the normal proton release group cannot deprotonate at this pH. 5) The time constant of early proton release is increased from 85 microseconds in the wild type to 1 ms in R82Q (in 150 mM salt). This can be directly attributed to the increase in the pKa of the proton release group and also explains the uncoupling of proton release from M formation. 6) In the E204Q mutant only late proton release is observed at both neutral and alkaline pH, consistent with the idea that E204 is the proton release group. The proton release is concurrent with the O-->bacteriorhodopsin transition, as in R82Q at neutral pH.  相似文献   

6.
Summary. Circular dichroism (CD) spectroscopy was employed for native (wild type, WT) bacteriorhodopsin (bR) and several mutant derivatives: R134K, R134H, R82Q, S35C, L66C, and R134C/E194C. Comparative analysis of the CD spectra in visible range shows that only R134C/E194C exhibits biphasic CD, typical for native bR, the other mutants demonstrate CD spectra with significantly smaller or absent negative band. Since the biphasic CD is a feature of hexagonal lattice structure composed by bR trimers in the purple membrane, these mutants and WT were examined by cross-linking studies, which confirmed the same trend towards trimeric organization. Therefore, a single amino acid substitution may lead to drastically different CD spectra without disruption of bR trimeric organization. Thus, although disruption of bR trimeric crystalline lattice structure (e.g., solubilization with detergents) directly results in the disappearance of characteristic bilobe in visible CD, the lack of the bilobe in the CD alone does not predict the absence of trimers.  相似文献   

7.
The redox potentials of the oriented films of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins (bR), prepared by adsorbing purple membrane (PM) sheets or its mutant on a Pt electrode, have been examined. The redox potentials (V) of the wild-type bR were -470 mV for the 13-cis configuration of the retinal Shiff base in bR and -757 mV for the all-trans configuration in H(2)O, and -433 mV for the 13-cis configuration and -742 mV for the all-trans configuration in D(2)O. The solvent isotope effect (DeltaV=V(D(2)O)-V(H(2)O)), which shifts the redox potential to a higher value, originates from the cooperative rearrangements of the extensively hydrogen-bonded water molecules around the protonated C=N part in the retinal Schiff base. The redox potential of bR was much higher for the 13-cis configuration than that for the all-trans configuration. The redox potentials for the E194Q mutant in the extracellular region were -507 mV for the 13-cis configuration and -788 mV for the all-trans configuration; and for the E204Q mutant they were -491 mV for the 13-cis configuration and -769 mV for the all-trans configuration. Replacement of the Glu(194) or Glu(204) residues by Gln weakened the electron withdrawing interaction to the protonated C=N bond in the retinal Schiff base. The E204 residue is less linked with the hydrogen-bonded network of the proton release pathway compared with E194. The redox potentials of the D96N mutant in the cytoplasmic region were -471 mV for the 13-cis configuration and -760 mV for the all-trans configuration which were virtually the same as those of the wild-type bR, indicating that the D to N point mutation of the 96 residue had no influence on the interaction between the D96 residue and the C=N part in the Schiff base under the light-adapted condition. The results suggest that the redox potential of bR is closely correlated to the hydrogen-bonded network spanning from the retinal Schiff base to the extracellular surface of bR in the proton transfer pathway.  相似文献   

8.
The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).  相似文献   

9.
The last stages of the photocycle of the photosynthetic pigment all-trans bacteriorhodopsin (bR570), as well as its proton pump mechanism, are markedly pH dependent. We have measured the relative amount of the accumulated O630 intermediate (Phir), as well as its rise and decay rate constants (kr and kd, respectively), over a wide pH range. The experiments were carried out in deionized membrane suspensions to which varying concentrations of metal cations and of large organic cations were added. The observed pH dependencies, s-shaped curves in the case of Phir and bell-shaped curves for kr and kd, are interpreted in terms of the titration of three protein residues denoted as R1, R2, and R3. The R1 titration is responsible for the increase in Phir, kr, and kd upon lowering the pH from pH approximately 9.5 to 7. At low pH Phir exhibits a secondary rise which is attributed to the titration of a low pKa group, R2. After reaching a maximum at pH approximately 7, kr and kd undergo a decrease upon decreasing the pH, which is attributed to the titration of R3. All three titrations exhibit pKa values which decrease upon increasing the salt concentration. As in the case of the Purple (bR570) if Blue (bR605) equilibrium, divalent cations are substantially more effective than monovalent cations in shifting the pKa values. Moreover, bulky organic cations are as effective as small metal cations. It is concluded that analogously to the Purple if Blue equilibrium, the salt binding sites which control the pKa values of R1, R2, and R3 are located on, or close to, the membrane surface. Possible identifications of the three protein residues are considered. Experiments with the E204Q mutant show that the mutation has markedly affected the R2 (Phir) titration, suggesting that R2 should be identified with Glu-204 or with a group whose pKa is affected by Glu-204. The relation between the R1, R2 and R3 titrations and the proton pump mechanism is discussed. It is evident that the pH dependence of Phir is unrelated to the measured pKa of the group (XH) which releases the proton to the extracellular medium during the photocycle. However, since the same residue may exhibit different pKa values at different stages of the photocycle, it cannot be excluded that R2 or R3 may be identified with XH.  相似文献   

10.
Sensory rhodopsin II (SRII) in Halobacterium salinarum membranes is a phototaxis receptor that signals through its bound transducer HtrII for avoidance of blue-green light. In the present study we investigated the proton movements during the photocycle of SRII in the HtrII-free and HtrII-complexed form. We monitored sustained light-induced pH changes with a pH electrode, and laser flash-induced pH changes with the pH indicator pyranine using sealed membrane vesicles and open sheets containing the free or the complexed receptor. The results demonstrated that SRII takes up a proton in M-to-O conversion and releases it during O-decay. The uptake and release are from and to the extracellular side, and therefore SRII does not transport the proton across the membrane. The pH dependence of the SRII photocycle indicated the presence of a protonatable group (pK(a) approximately 7.5) in the extracellular proton-conducting path, which plays a role in proton uptake by the Schiff base in the M-to-O conversion. The extracellular proton circulation produced by SRII was not blocked by HtrII complexation, unlike the cytoplasmic proton conduction in SRI that was found in the same series of measurements to be blocked by its transducer, HtrI. The implications of this finding for current models of SRI and SRII signaling are discussed.  相似文献   

11.
The pK(a) values of D85 in the wild-type and R82Q, as well as R82A recombinant bacteriorhodopsins, and the Schiff base in the D85N, D85T, and D85N/R82Q proteins, have been determined by spectroscopic titrations in the dark. They are used to estimate the coulombic interaction energies and the pK(a) values of the Schiff base, D85, and R82 during proton transfer from the Schiff base to D85, and the subsequent proton release to the bulk in the initial part of the photocycle. The pK(a) of the Schiff base before photoexcitation is calculated to be in effect only 5.3-5.7 pH units higher than that of D85; overcoming this to allow proton transfer to D85 requires about two thirds of the estimated excess free energy retained after absorption of a photon. The proton release on the extracellular surface is from an unidentified residue whose pK(a) is lowered to about 6 after deprotonation of the Schiff base (Zimanyi, L., G. Varo, M. Chang, B. Ni, R. Needleman, and J.K. Lanyi, 1992. Biochemistry. 31:8535-8543). We calculate that the pK(a) of the R82 is 13.8 before photoexcitation, and it is lowered after proton exchange between the Schiff base and D85 only by 1.5-2.3 pH units. Therefore, coulombic interactions alone do not appear to change the pK(a) of R82 as much and D85 only by 1.5-2.3 pH units. Therefore, coulombic interactions alone do not appear to change the pK(a) of R82 as much as required if it were the proton release group.  相似文献   

12.
Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 and Glu204, two amino acids that are involved in proton release to the extracellular side of bacteriorhodopsin. Difference spectra of films of E194Q, E204Q, E194Q/E204Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q at 243, 277, and 293 K and several pH values were obtained by continuous illumination. A specific effect of Cl(-) ions was found for the mutants, promoting a N-like intermediate at alkaline pH and an O' intermediate at neutral or acid pH. The apparent pK(a) of Asp85 in the M intermediate was found to be decreased for E194Q in the presence of Cl(-) (pK(a) of 7.6), but it was unchanged for E204Q, as compared to wild-type. In the absence of Cl(-) (i.e., in the presence of SO(4)(2)(-)), mutation of Glu194 or of Glu204 produces M- (or M(N), M(G))-like intermediates under all of the conditions examined. The absence of N, O, and O' intermediates suggests a long-range effect of the mutation. Furthermore, it is suggested that Cl(-) acts by reaching the interior of the protein, rather than producing surface effects. The effect of low water content was also examined, in the presence of Cl(-). Similar spectra corresponding to the M(1) intermediate were found for dry samples of both mutants, indicating that the effects of the mutations or of Cl(-) ions are confined to the second part of the photocycle. The water O-H stretching data further confirms altered photocycles and the effect of Cl(-) on the accumulation of the N intermediate.  相似文献   

13.
The redox potentials of the oriented films of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins (bR), prepared by adsorbing purple membrane (PM) sheets or its mutant on a Pt electrode, have been examined. The redox potentials (V) of the wild-type bR were −470 mV for the 13-cis configuration of the retinal Shiff base in bR and −757 mV for the all-trans configuration in H2O, and −433 mV for the 13-cis configuration and −742 mV for the all-trans configuration in D2O. The solvent isotope effect (ΔV=V(D2O)−V(H2O)), which shifts the redox potential to a higher value, originates from the cooperative rearrangements of the extensively hydrogen-bonded water molecules around the protonated CN part in the retinal Schiff base. The redox potential of bR was much higher for the 13-cis configuration than that for the all-trans configuration. The redox potentials for the E194Q mutant in the extracellular region were −507 mV for the 13-cis configuration and −788 mV for the all-trans configuration; and for the E204Q mutant they were −491 mV for the 13-cis configuration and −769 mV for the all-trans configuration. Replacement of the Glu194 or Glu204 residues by Gln weakened the electron withdrawing interaction to the protonated CN bond in the retinal Schiff base. The E204 residue is less linked with the hydrogen-bonded network of the proton release pathway compared with E194. The redox potentials of the D96N mutant in the cytoplasmic region were −471 mV for the 13-cis configuration and −760 mV for the all-trans configuration which were virtually the same as those of the wild-type bR, indicating that the D to N point mutation of the 96 residue had no influence on the interaction between the D96 residue and the CN part in the Schiff base under the light-adapted condition. The results suggest that the redox potential of bR is closely correlated to the hydrogen-bonded network spanning from the retinal Schiff base to the extracellular surface of bR in the proton transfer pathway.  相似文献   

14.
At high pH (> 8) the 570 nm absorption band of all-trans bacteriorhodopsin (bR) in purple membrane undergoes a small (1.5 nm) shift to longer wavelengths, which causes a maximal increase in absorption at 615 nm. The pK of the shift is 9.0 in the presence of 167 mM KCl, and its intrinsic pK is ~8.3. The red shift of the trans-bR absorption spectrum correlates with the appearance of the fast component in the light-induced L to M transition, and absorption increases at 238 and 297 nm which are apparently caused by the deprotonation of a tyrosine residue and red shift of the absorption of tryptophan residues. This suggests that the deprotonation of a tyrosine residue with an exceptionally low pK (pKa ≈ 8.3) is responsible for the absorption shift of the chromophore band and fast M formation. The pH and salt dependent equilibrium between the two forms of bR, “neutral” and “alkaline,” bR ↔ bRa, results in two parallel photocycles of trans-bR at high pH, differing in the rate of the L to M transition. In the pH range 10-11.8 deprotonation of two more tyrosine residues is observed with pK's ~ 10.3 and 11.3 (in 167 mM KCL). Two simple models discussing the role of the pH induced tyrosine deprotonation in the photocycle and proton pumping are presented.

It is suggested that the shifts of the absorption bands at high pH are due to the appearance of a negatively charged group inside the protein (tyrosinate) which causes electrochromic shifts of the chromophore and protein absorption bands due to the interaction with the dipole moments in the ground and excited states of bR (Stark effect). This effect gives evidence for a significant change in the dipole moment of the chromophore of bR upon excitation.

Under illumination alkaline bR forms, besides the usual photocycle intermediates, a long-lived species with absorption maximum at 500 nm (P500). P500 slowly converts into bRa in the dark. Upon illumination P500 is transformed into an intermediate having an absorption maximum at 380 nm (P380). P380 can be reconverted to P500 by blue light illumination or by incubation in the dark.

  相似文献   

15.
Zadok U  Asato AE  Sheves M 《Biochemistry》2005,44(23):8479-8485
The retinal protein protonated Schiff base linkage plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment, the Schiff base (SB) is titrated with a pK(a) of approximately 13, but following light absorption, it experiences a decrease in the pK(a) and undergoes several alterations, including a deprotonation process. We have studied the SB titration using retinal analogues which have intrinsically lower pK(a)'s which allow for SB titrations over a much lower pH range. We found that above pH 9 the channel for the SB titration is perturbed, and the titration rate is considerably reduced. On the basis of studies with several mutants, it is suggested that the protonation state of residue Glu204 is responsible for the channel perturbation. We suggest that above pH 12 a channel for the SB titration is restored probably due to titration of an additional protein residue. The observations may imply that during the bR photocycle and M photointermediate formation the rate of Schiff base protonation from the bulk is decreased. This rate decrease may be due to the deprotonation process of the "proton-releasing complex" which includes Glu204. In contrast, during the lifetime of the O intermediate, the protonated SB is exposed to the bulk. Possible implications for the switch mechanism, and the directionality of the proton movement, are discussed.  相似文献   

16.
The kinetics of the photoreversal reaction of the M-intermediate of bacteriorhodopsin (bR) was investigated by time-resolved optical absorption spectroscopy and photovoltage measurements using double-flash excitation (a green flash (532 nm) followed by a blue flash (400 nm) after a variable delay). The sign of the photovoltage and the 1H/2H kinetic isotope effect indicate that the Schiff base is reprotonated by a group between the Schiff base and the extracellular surface, probably Asp85. Analysis of the kinetic data shows that the charge movement in 150 mM KCl at 12 degrees C is characterized by two components with time constants of approximately 100 ns and approximately 600 ns, respectively, which are independent of the delay time between the flashes and the pH. The amplitudes of the fast and slow components depend on the delay and the pH. The slower component starts to contribute to the charge movement only after delays longer than 100 micros, is absent at low pH, and increases in amplitude with a pKa of approximately 6. Because the proton release group deprotonates after 70-100 micros and has a transient pKa of 5.8, these results suggest the following assignment: the fast and the combination of fast and slow components represent photoreversal from two M states, with the release group protonated and deprotonated, respectively. The slow phase of the photoreversal starts from a state with the release group deprotonated, and with the pK of Asp85 elevated, and is probably due to the restoration of the pK of Asp85 to its initial low value. This provides further evidence for coupling between the pK's of Asp85 and the release group and suggests that proton release is the first step in the reprotonation switch. At alkaline pH the amplitude of the electrical signal from the back photoreaction decreases with an apparent pK of 8, without a corresponding decrease in the amount of M. At neutral pH the movement of the positively charged guanidinium group of Arg82 from a position near the release group on the surface to Asp85 makes a substantial contribution to the electrical photoreversal amplitude. Above the pK of the release group in the unphotolysed state (approximately 8), Arg82 stays near the surface, leading to a corresponding signal reduction.  相似文献   

17.
The contribution of proton release from the so-called proton release group to the microsecond B2 photocurrent from bacteriorhodopsin (bR) oriented in polyacrylamide gels was determined. The fraction of the B2 current due to proton release was resolved by titration of the proton release group in M. At pH values below the pKa of the proton release group in M, the proton release group cannot release its proton during the first half of the bacteriorhodopsin photocycle. At these pH values, the B2 photocurrent is due primarily to translocation of the Schiff base proton to Asp85. The B2 photocurrent was measured in wild-type bR gels at pH 4.5-7.5, in 100 mM KCl/50 mM phosphate. The B2 photocurrent area (proportional to the amount of charge moved) exhibits a pH dependence with a pKa of 6.1. This is suggested to be the pKa of the proton release group in M; the value obtained is in good agreement with previous results obtained by examining photocycle kinetics and pH-sensitive dye signals. In the mutant Glu204Gln, the B2 photocurrent of the mutant membranes was pH independent between pH 4 and 7. Because the proton release group is incapacitated, and early proton release is eliminated in the Glu204Gln mutant, this supports the idea that the pH dependence of the B2 photocurrent in the wild type reflects the titration of the proton release group. In wild-type bacteriorhodopsin, proton release contributes approximately half of the B2 area at pH 7.5. The B2 area in the Glu204Gln mutant is similar to that in the wild type at pH 4.5; in both cases, the B2 current is likely due only to movement of the Schiff base proton to Asp85.  相似文献   

18.
The photoreaction of the E194Q mutant of bacteriorhodopsin has been investigated at various pH values by time-resolved step-scan Fourier-transform infrared difference spectroscopy employing the attenuated total reflection technique. The difference spectrum at pH 8.4 is comparable to the N-BR difference spectra of the wild type with the remarkable exception that D85 is deprotonated. Since the retinal configuration is not perturbed by the E194Q mutation, it is concluded that there is no interaction of D85 with retinal during the lifetime of the N state. At pH 6, a consecutive state to the O intermediate is detected in which D212 is transiently protonated. The comparison with wild-type bacteriorhodopsin reveals that protonation of D212 represents an intermediate step during proton transfer from D85 to the proton release group in the final stage of the reaction cycle. The described effects are more pronounced in the E194Q mutant than in the E204Q mutant demonstrating different roles of these two glutamates/glutamic acids at least in the final stages of the catalytic cycle of bacteriorhodopsin.  相似文献   

19.
Calculations of protonation states and pK(a) values for the ionizable groups in the resting state of bacteriorhodopsin have been carried out using the recently available 1.55 A resolution X-ray crystallographic structure. The calculations are in reasonable agreement with the available experimental data for groups on or near the ion transport chain (the retinal Schiff base; Asp85, 96, 115, 212, and Arg82). In contrast to earlier studies using lower-resolution structural data, this agreement is achieved without manipulations of the crystallographically determined heavy-atom positions or ad hoc adjustments of the intrinsic pK(a) of the Schiff base. Thus, the theoretical methods used provide increased reliability as the input structural data are improved. Only minor effects on the agreement with experiment are found with respect to methodological variations, such as single versus multi-conformational treatment of hydrogen atom placements, or retaining the crystallographically determined internal water molecules versus treating them as high-dielectric cavities. The long-standing question of the identity of the group that releases a proton to the extracellular side of the membrane during the L-to-M transition of the photocycle is addressed by including as pH-titratable sites not only Glu204 and Glu194, residues near the extracellular side that have been proposed as the release group, but also an H(5)O(2)(+) molecule in a nearby cavity. The latter represents the recently proposed storage of the release proton in an hydrogen-bonded water network. In all calculations where this possibility is included, the proton is stored in the H(5)O(2)(+) rather than on either of the glutamic acids, thus establishing the plausibility on theoretical grounds of the storage of the release proton in bacteriorhodopsin in a hydrogen-bonded water network. The methods used here may also be applicable to other proteins that may store a proton in this way, such as the photosynthetic reaction center and cytochrome c oxidase.  相似文献   

20.
Song Y  Mao J  Gunner MR 《Biochemistry》2003,42(33):9875-9888
Residue ionization states were calculated in nine crystal structures of bacteriorhodopsin trapped in bR, early M, and late M states by multiconformation continuum electrostatics. This combines continuum electrostatics and molecular mechanics, deriving equilibrium distributions of ionization states and polar residue and water positions. The three central cluster groups [retinal Schiff base (SB), Asp 85 and Asp 212] are ionized in bR structures while a proton has transferred from SB(+) to Asp 85(-) in late M structures matching experimental results. The proton shift in M is due to weaker SB(+)-ionized acid and more favorable SB(0)-ionized acid interactions following retinal isomerization. The proton release cluster (Glu 194 and Glu 204) binds one proton in bR, which is lost to water by pH 8 in late M. In bR the half-ionized state is stabilized by charge-dipole interactions while full ionization is disallowed by charge-charge repulsion between the closely spaced acids. In M the acids move apart, permitting full ionization. Arg 82 movement connects the proton shifts in the central and proton release clusters. Changes in total charge of the two clusters are coupled by direct long-range interactions. Separate calculations consider continuum or explicit water in internal cavities. The explicit waters and nearby polar residues can reorient to stabilize different charge distributions. Proton release to the low-pH, extracellular side of the protein occurs in these calculations where residue ionization remains at equilibrium with the medium. Thus, the key changes distinguishing the intermediates are indeed trapped in the structures.  相似文献   

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